NZ749239A

NZ749239A - Method for producing dna probe and method for analyzing genomic dna using the dna probe

Info

Publication number: NZ749239A
Application number: NZ749239A
Authority: NZ
Inventors: Hiroyuki Enoki; Minoru Inamori; Yoshie Takeuchi
Original assignee: Toyota Jidosha Kabushiki Kaisha
Priority date: 2016-06-29
Filing date: 2017-06-26

Abstract

This invention provides a DNA probe that is applicable to a DNA library prepared in a simple manner with excellent reproducibility. Such DNA probe is produced by a method comprising steps of performing a nucleic acid amplification reaction in a reaction solution containing genomic DNA and a random primer at a high concentration, so as to obtain a DNA fragments with the use of the genomic DNA as a template; determining the nucleotide sequence of the resulting DNA fragments; and, on the basis of the nucleotide sequence of the DNA fragments obtained in the step above, designing a DNA probe used for detecting a DNA fragment. rimer at a high concentration, so as to obtain a DNA fragments with the use of the genomic DNA as a template; determining the nucleotide sequence of the resulting DNA fragments; and, on the basis of the nucleotide sequence of the DNA fragments obtained in the step above, designing a DNA probe used for detecting a DNA fragment.

Description

Description Title of Invention: METHOD FOR PRODUCING DNA PROBE AND METHOD FOR ANALYZING GENOMIC DNA USING THE DNA PROBE Technical Field The present invention relates to a method for producing a DNA library that can be used for analyzing a DNA marker or other purposes and a method for gene analysis using such DNA library.

Background Art In general, genomic is is performed to conduct comprehensive analysis of genetic information contained in the genome, such as nucleotide sequence information. r, an analysis aimed at determination of the nucleotide sequence for whole genome is disadvantageous in terms of the number of processes and the cost. In cases of organisms with large genomic sizes, in addition, genomic analysis based on nu— cleotide sequence analysis has tions because of genome complexity.

Patent Literature 1 discloses an amplified fragment length polymorphism (AFLP) marker technique wherein a —specific marker is orated into a restriction— enzyme—treated fragment that had been ligated to an adaptor and only a part of the sequence of the restriction—enzyme—treated fragment is to be determined. According to the technique disclosed in Patent Literature 1, the complexity of c DNA is reduced by treating genomic DNA with a restriction enzyme, the tide sequence of a target part of the restriction—enzyme—treated fragment is ined, and the target restriction—enzyme—treated nt is thus determined sufficiently. The technique disclosed in Patent Literature 1, however, requires processes such as treatment of genomic DNA with a restriction enzyme and ligation reaction with the use of an r. Thus, it is difficult to achieve a cost reduction.

Meanwhile, Patent Literature 2 discloses as follows. That is, a DNA marker for iden— tification that is highly correlated with the s of taste tion was found from among DNA bands obtained by amplifying DNAs extracted from a rice sample via PCR in the presence of adequate primers by the so—called RAPD (randomly amplified polymorphic DNA) technique. The method disclosed in Patent Literature 2 involves the use of a plurality of sequence—tagged sites (STSs, which are primers) identified by particular sequences. According to the method disclosed in Patent Literature 2, a DNA marker for identification ied with the use of an STS primer is detected via elec— trophoresis. However, the RAPD technique disclosed in Patent Literature 2 yields sig— nificantly poor reproducibility of PCR amplification, and, accordingly, such technique cannot be generally adopted as a DNA marker technique.

Patent ture 3 discloses a method for producing a genomic library wherein PCR is carried out with the use of a single type of primer designed on the basis of a sequence that appears relatively frequently in the target genome, the entire c region is substantially mly amplified, and a c library can be thus produced. While Patent Literature 3 describes that a genomic library can be ed by conducting PCR with the use of a random primer containing a random sequence, it does not describe any actual procedures or results of experimentation. Accordingly, the method described in Patent Literature 3 is d to require nucleotide sequence in— formation of the genome so as to identify the genome appearing frequency, which would increase the number of procedures and the cost. According to the method described in Patent Literature 3, in addition, the entire genome is to be amplified, and complexity of genomic DNA cannot be reduced, disadvantageously.

Patent ture 4 discloses a high—throughput technique associated with markers that involves reduction in genome complexity by restriction enzyme treatment in com— bination with an array technique. According to the technique associated with markers sed in Patent ture 4, genomic DNA is digested with restriction enzymes, an adaptor is ligated to the resulting genomic DNA fragment, a DNA fragment is amplified with the use of a primer hybridizing to the adaptor, and a DNA probe used for detection of such DNA fragment is then designed on the basis of the nucleotide ce of the amplified DNA fragment.

In addition, Non—Patent ture 1 discloses the development of high—density linkage map containing several thousands of DNA markers for sugarcane and wheat by making use of the technique disclosed in Patent ture 4. Also, Non—Patent Literature 2 ses the development of a high—density e map containing several thousands of DNA markers for buck wheat by making use of the technique disclosed in Patent Literature 4.

Further, Patent Literature 5 discloses a method involving the use of a random primer as a sample to be reacted with an array on which a probe is immobilized. However, Patent Literature 5 does not discloses a method in which a random primers is used to obtain an amplified fragment and the resulting amplified fragment is used to construct a DNA library.

Citation List Patent Literature PTL 1: JP Patent No. 5389638 PTL 2: JP 2003—79375 A PTL 3: JP Patent No. 3972106 PTL 4: JP Patent No. 5799484 PTL 5: JP 2014—204730 A Non Patent Literature NPL 1: DNA Research 21, 555—567, 2014 NPL 2: Breeding Science 64: 291—299, 2014 y of ion Technical Problem A technique for genome information analysis, such as genetic linkage is conducted with the use of a DNA marker, is desired to produce a DNA library in a more convenient and highly ucible manner. In addition, such technique is desired to produce a DNA probe capable of ing a DNA fragment ned in a DNA library with high accuracy. As described above, a wide variety of techniques for producing a DNA library and a DNA probe are known. To date, however, there have been no techniques known to be ient in terms of ience and/or repro— ducibility. Under the above circumstances, it is an object of the present invention to provide a method for producing a DNA probe that is applicable to a DNA library produced by a method with more convenience and higher reproducibility, and it is another object to provide a method for analyzing genomic DNA with the use of such DNA probe.

Solution to m The t inventors have conducted concentrated studies in order to attain the above objects. As a result, they discovered that a DNA library could be produced with high reproducibility by conducting PCR with the use of a random primer while des— ignating the concentration of such random primer within a designated range in a reaction solution and that a DNA probe could be easily designed on the basis of the nu— cleotide sequences of the DNA library to be produced. This has led to the completion of the present invention.

The present invention includes the following. (1) A method for producing a DNA probe comprising steps of: conducting a c acid amplification reaction in a reaction solution containing genomic DNA and a random primer at a high concentration using genomic DNA as a template to obtain DNA fragments; determining the nucleotide sequences of the obtained DNA fragments; and designing a DNA probe used for ing a DNA fragment obtained in the above step on the basis of the nucleotide sequences of such DNA fragments. (2) The method for producing a DNA probe according to (1), wherein DNA fragments are ed from a plurality of different genomic DNAs with the use of the random primers and, on the basis of the nucleotide sequences of the DNA fragments, the DNA probe containing regions different n such genomic DNAs is designed. (3) The method for producing a DNA probe according to (1), wherein the nucleotide sequence of the DNA fragment is compared with a known nucleotide sequence and the DNA probe containing a region different from that of the known nucleotide sequence is designed. (4) The method for producing a DNA probe ing to (1), wherein the reaction solution ns a random primer at a concentration of 4 to 200 microM. (5) The method for producing a DNA probe ing to (1), wherein the reaction solution ns a random primer at a tration of 4 to 100 microM. (6) The method for producing a DNA probe according to (1), n the random primers each contain 9 to 30 nucleotides. (7) The method for producing a DNA probe according to (1), wherein the DNA fragments contain 100 to 500 nucleotides. (8) A method for analyzing genomic DNA comprising steps of: bringing the DNA probe produced by the method for producing a DNA probe according to any of (l) to (7) into contact with a DNA fragment derived from genomic DNA subjected to analysis; and detecting hybridization occurring between the DNA probe and the DNA fragment. (9) The method for analyzing genomic DNA ing to (8), which further comprises a step of conducting a nucleic acid amplification reaction with the use of the genomic DNA subjected to analysis and the random primer to obtain the DNA fragment. (10) The method for analyzing genomic DNA according to (8), wherein the DNA fragment derived from genomic DNA is a DNA marker and the presence or absence of the DNA marker is detected with the use of the DNA probe. (1 1) An apparatus for DNA analysis comprising the DNA probe produced by the method for producing a DNA probe according to any of (l) to (7) and a support sing the DNA probe immobilized thereon. (12) The apparatus for DNA analysis according to (l 1), wherein the support is a substrate or bead.

Advantageous s of Invention In the method for ing a DNA probe according to the present invention, a nu— cleotide sequence of a DNA probe is designed based on the nucleotide sequence of DNA fragments produced by the method of nucleic acid amplification using a random primer at a high concentration. According to the method of nucleic acid amplification using a random primer at a high concentration, DNA fragments can be amplified with excellent ucibility. ing to the present invention, therefore, a DNA probe applicable to a DNA fragment that can be obtained while achieving excellent repro— lity can be produced in a simple manner.

According to the method for producing a DNA probe according to the present invention, also, a DNA probe applicable to a DNA fragment can be produced while achieving excellent reproducibility, and the resulting DNA probe can be used for genetic analysis, such as genetic linkage is, ing the use of a DNA nt of interest as a DNA marker.

The method for analyzing genomic DNA with the use of a DNA probe according to the present invention involves the use of a DNA probe applicable to a DNA fragment produced in a simple manner with excellent reproducibility. Accordingly, genomic DNA can be ed in a cost—effective manner with high accuracy.

Brief Description of Drawings [fig.1]Fig. 1 shows a ﬂow chart demonstrating a method for producing a DNA library and a method for genetic analysis with the use of the DNA library. [fig.2]Fig. 2 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library ied via PCR using DNA of the sugarcane variety NiFS as a template under general conditions. [fig.3]Fig. 3 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template at an annealing tem— perature of 45 degrees C. [fig.4]Fig. 4 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template at an annealing tem— perature of 40 degrees C. [fig.5]Fig. 5 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic n of the DNA library amplified using DNA of the ane variety NiFS as a template at an annealing tem— perature of 37 degrees C.

]Fig. 6 shows a characteristic diagram demonstrating a ation between an ied nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the ane variety NiFS as a template and 2.5 units of an enzyme. [fig.7]Fig. 7 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA y amplified using DNA of the ane y NiFS as a template and 12.5 units of an enzyme. [fig.8]Fig. 8 shows a characteristic diagram trating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and MgClz at the concentration doubled from the original level.

]Fig. 9 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and MgClz at the concentration tripled from the al level. [fig. lO]Fig. 10 shows a characteristic diagram trating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and MgClz at the concentration quadrupled concentration. [fig. 1 l]Fig. ll shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 8 bases. [fig.12]Fig. 12 shows a characteristic diagram trating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 9 bases. [fig. l3]Fig. 13 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising ll bases. [fig. l4]Fig. 14 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a te and a random primer comprising 12 bases. [fig.15]Fig. 15 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 14 bases. [fig.16]Fig. 16 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 16 bases. [fig.17]Fig. 17 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 18 bases. [fig.18]Fig. 18 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 20 bases. [fig.19]Fig. 19 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a te and a random primer at a concentration of 2 microM. 0]Fig. 20 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer at a concentration of 4 . [fig.21]Fig. 21 shows a teristic m demonstrating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 6 microM. [fig.22]Fig. 22 shows a characteristic m demonstrating a correlation n an amplified nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic n (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 6 microM. [fig.23]Fig. 23 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 8 microM. [fig.24]Fig. 24 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 8 microM. [fig.25]Fig. 25 shows a teristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 10 microM. [fig.26]Fig. 26 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic n (appeared for the second time) of the DNA y amplified using DNA of the sugarcane y NiFS as a template and a random primer at a concentration of 10 microM. [fig.27]Fig. 27 shows a characteristic diagram trating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern red for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 20 microM. [fig.28]Fig. 28 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 20 microM. 9]Fig. 29 shows a characteristic diagram demonstrating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern (appeared for the WO 03727 first time) of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer at a concentration of 40 microM. [fig.30]Fig. 30 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 40 microM. l]Fig. 31 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a tration of 60 microM. [fig.32]Fig. 32 shows a teristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer at a concentration of 60 microM. [fig.33]Fig. 33 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 100 microM. [fig.34]Fig. 34 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 100 .

]Fig. 35 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA y amplified using DNA of the sugarcane variety NiFS as a te and a random primer at a concentration of 200 . [fig.36]Fig. 36 shows a characteristic diagram demonstrating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an ophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 200 microM. [fig.37]Fig. 37 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library ied using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 300 microM. [fig.38]Fig. 38 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and a random primer at a concentration of 300 . [fig.39]Fig. 39 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and a random primer at a concentration of 400 microM. [fig.40]Fig. 40 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern red for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a tration of 400 . [fig.4l]Fig. 41 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a cence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and a random primer at a concentration of 500 microM. [fig.42]Fig. 42 shows a teristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a te and a random primer at a concentration of 500 microM. [fig.43]Fig. 43 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 600 microM. [fig.44]Fig. 44 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 700 microM. [fig.45]Fig. 45 shows a teristic diagram demonstrating a correlation n an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer at a concentration of 800 microM. [fig.46]Fig. 46 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a cence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer at a concentration of 900 microM. [fig.47]Fig. 47 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer at a concentration of 1000 microM. [fig.48]Fig. 48 shows a characteristic diagram demonstrating the results of MiSeq is of a DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer. [fig.49]Fig. 49 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is ined based on an electrophoretic pattern (appeared for the first time) of the DNA library ied using DNA of the rice variety Nipponbare as a template and a random primer. [fig.50]Fig. 50 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the rice variety Nipponbare as a template and a random . [fig.51]Fig. 51 shows a characteristic diagram trating the results of MiSeq analysis of a DNA library amplified using DNA of the rice variety Nipponbare as a template and a random primer. [fig.52]Fig. 52 shows a characteristic diagram demonstrating positions of MiSeq read patterns in the genome information of the rice variety bare. [fig.53]Fig. 53 shows a characteristic diagram demonstrating the frequency distribution of the number of mismatched nucleotides between the random primer and the rice genome. [fig.54]Fig. 54 shows a teristic diagram demonstrating the number of reads of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the marker N80521 152. [fig.55]Fig. 55 shows a photograph demonstrating e1ectrophoretic patterns of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the PCR marker N80521 152. [fig.56]Fig. 56 shows a characteristic diagram demonstrating the number of reads of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the marker N80997 192. [fig.57]Fig. 57 shows a photograph demonstrating ophoretic patterns of the ane varieties NiFS and Ni9 and hybrid progeny lines thereof at the PCR marker N80997 192. [fig.58]Fig. 58 shows a characteristic diagram trating the number of reads of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines f at the marker N80533 142. [fig.59]Fig. 59 shows a photograph demonstrating ophoretic patterns of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the PCR marker N80533 142. [fig.60]Fig. 60 shows a characteristic diagram demonstrating the number of reads of the ane ies NiFS and Ni9 and hybrid progeny lines thereof at the marker N9 155239 1 . [fig.61]Fig. 61 shows a photograph demonstrating e1ectrophoretic patterns of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the PCR marker N9 155239 1 . [fig.62]Fig. 62 shows a characteristic diagram demonstrating the number of reads of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the marker N9 1653962. [fig.63]Fig. 63 shows a photograph demonstrating e1ectrophoretic patterns of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the PCR marker N9 2. [fig.64]Fig. 64 shows a characteristic diagram demonstrating the number of reads of the sugarcane varieties NiFS and Ni9 and hybrid progeny lines thereof at the marker N91 124801. [fig.65]Fig. 65 shows a photograph demonstrating e1ectrophoretic patterns of the sugarcane varieties NiFS and Ni9 and hybrid y lines thereof at the PCR marker N91 124801. [fig.66]Fig. 66 shows a characteristic diagram demonstrating a correlation n an amplified fragment length and a cence unit (FU) in which the amplified fragment length is determined based on an e1ectrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 9 bases. [fig.67]Fig. 67 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a cence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the ane variety NiFS as a template and a random primer comprising 9 bases. 8]Fig. 68 shows a characteristic diagram trating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern red for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 10 bases. [fig.69]Fig. 69 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 10 bases. [fig.70]Fig. 70 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 11 bases. [fig.7l]Fig. 71 shows a teristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied nt length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer comprising 11 bases. [fig.72]Fig. 72 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and a random primer comprising 12 bases. [fig.73]Fig. 73 shows a characteristic diagram demonstrating a ation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and a random primer comprising 12 bases. [fig.74]Fig. 74 shows a characteristic diagram demonstrating a ation between an W0 2018/003727 amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic n (appeared for the first time) of the DNA library amplified using DNA of the ane variety NiF8 as a template and a random primer comprising 14 bases. [fig.75]Fig. 75 shows a characteristic diagram demonstrating a correlation between an amplified nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer sing 14 bases. [fig.76]Fig. 76 shows a characteristic m demonstrating a correlation n an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 16 bases. [fig.77]Fig. 77 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 16 bases. [fig.78]Fig. 78 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified nt length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 18 bases. [fig.79]Fig. 79 shows a characteristic diagram demonstrating a correlation between an ied nt length and a cence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 18 bases. 0]Fig. 80 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 20 bases. [fig.81]Fig. 81 shows a teristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiF8 as a template and a random primer comprising 20 bases. [fig.82]Fig. 82 shows a characteristic diagram demonstrating the results of inves— tigating the reproducibility of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and random primers each comprising 8 to 35 bases used at a concentration of 0.6 to 300 microM. [fig.83]Fig. 83 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified nt length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a single type of random primer. [fig.84]Fig. 84 shows a characteristic diagram trating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the ane variety NiFS as a template and a single type of random primer. [fig.85]Fig. 85 shows a characteristic diagram trating a correlation between an amplified fragment length and a cence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the ane variety NiFS as a template and 2 types of random primers. [fig.86]Fig. 86 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library ied using DNA of the sugarcane variety NiFS as a template and 2 types of random primers. [fig.87]Fig. 87 shows a characteristic diagram demonstrating a correlation between an amplified nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic n (appeared for the first time) of the DNA library amplified using DNA of the ane variety NiFS as a template and 3 types of random primers. [fig.88]Fig. 88 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and 3 types of random primers. [fig.89]Fig. 89 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic n (appeared for the first time) of the DNA library amplified using DNA of the sugarcane y NiFS as a template and 12 types of random primers. [fig.90]Fig. 90 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the ane variety NiFS as a template and 12 types of random primers. [fig.91]Fig. 91 shows a characteristic diagram demonstrating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library ied using DNA of the sugarcane variety NiFS as a template and 24 types of random primers. [fig.92]Fig. 92 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the ied fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the ane variety NiFS as a template and 24 types of random primers. [fig.93]Fig. 93 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a cence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and 48 types of random s. 4]Fig. 94 shows a characteristic m demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and 48 types of random primers. [fig.95]Fig. 95 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer B comprising 10 nucleotides. [fig.96]Fig. 96 shows a characteristic diagram demonstrating a correlation between an amplified nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an ophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer B comprising 10 nucleotides. [fig.97]Fig. 97 shows a characteristic diagram demonstrating a ation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified nt length is determined based on an ophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer C comprising 10 nucleotides. [fig.98]Fig. 98 shows a characteristic diagram demonstrating a correlation between an amplified nt length and a ﬂuorescence unit (FU) in which the amplified fragment length is ined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer C comprising 10 nucleotides. [fig.99]Fig. 99 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a te and a random primer D comprising 10 nucleotides. [fig. lOO]Fig. 100 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a random primer D comprising 10 nucleotides. [fig. lOl]Fig. 101 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an ophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer E comprising 10 nucleotides. 02]Fig. 102 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library ied using DNA of the sugarcane variety NiFS as a te and a random primer E comprising 10 tides. [fig. lO3]Fig. 103 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using DNA of the sugarcane variety NiFS as a template and a random primer F sing 10 nucleotides. [fig. lO4]Fig. 104 shows a characteristic diagram demonstrating a correlation between an ied fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern red for the second time) of the DNA y amplified using DNA of the sugarcane variety NiFS as a template and a random primer F comprising 10 nucleotides. [fig.105]Fig. 105 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the first time) of the DNA library amplified using human genomic DNA as a template and a random primer A comprising 10 nucleotides. [fig.106]Fig. 106 shows a characteristic diagram demonstrating a correlation between an amplified fragment length and a ﬂuorescence unit (FU) in which the amplified fragment length is determined based on an electrophoretic pattern (appeared for the second time) of the DNA library amplified using human genomic DNA as a template and a random primer A comprising 10 nucleotides. [fig.107]Fig. 107 shows a ﬂow chart demonstrating a s for ing a DNA rray with the application of the method for producing a DNA probe according to the present invention. [fig.108]Fig. 108 shows a characteristic diagram demonstrating the results of assaying signals obtained from a DNA probe concerning the DNA library amplified using genomic DNAs of NiFS and Ni9 as templates and a random primer at a high con— centration. [fig.109]Fig. 109 shows a characteristic diagram demonstrating the results of comparison of signals ed through repeated measurements concerning the DNA library amplified using c DNA of Ni9 as a template and a random primer at a high concentration. [fig.110]Fig. 110 shows a teristic diagram demonstrating the results of assaying signal levels obtained from the DNA probe ng with the marker N80521152. 11]Fig. 111 shows a characteristic diagram demonstrating the results of assaying signal levels obtained from the DNA probe reacting with the marker N80997192. [fig.112]Fig. 112 shows a characteristic diagram demonstrating the s of assaying signal levels obtained from the DNA probe ng with the marker N80533142. [fig.113]Fig. 113 shows a teristic diagram demonstrating the s of assaying signal levels obtained from the DNA probe reacting with the marker N91552391. [fig.114]Fig. 114 shows a characteristic diagram demonstrating the results of assaying signal levels ed from the DNA probe reacting with the marker N91653962. [fig.115]Fig. 115 shows a characteristic diagram demonstrating the results of assaying signal levels obtained from the DNA probe reacting with the marker N91124801.

Description of Embodiments Hereafter, the present invention is bed in detail.

According to the method for producing a DNA probe of the present invention, a nucleic acid amplification on is carried out in a reaction solution, which is prepared to n a primer having an arbitrary tide sequence (hereafter, referred to as a "random primer") at a high concentration, and a nucleotide ce of a DNA probe used for detecting an amplified nucleic acid fragment (i.e., a DNA fragment) is designed based on the nucleotide sequence of such DNA fragment. By conducting a nucleic acid amplification reaction in a reaction solution containing a random primer at a high concentration, a DNA fragment of interest can be ied with excellent reproducibility. Hereafter, the obtained DNA fragment is referred to as a "DNA library." When a reaction solution contains a random primer at a high concentration, such con— centration is higher than the concentration of a primer used in a l c acid amplification reaction. When producing a DNA library, ically, a random primer is used at a higher concentration than a primer used in a general nucleic acid ampli— fication reaction. As a template contained in a reaction solution, genomic DNA prepared from a target organism for which a DNA library is to be ed can be used. A target organism species is not particularly limited, and a target organism species can be, for example, an animal including a human, a plant, a microorganism, or a virus. That is, a DNA library can be produced from any organism species.

When producing a DNA library, the concentration of a random primer may be prescribed as described above. Thus, a nucleic acid fragment (or nucleic acid fragments) can be amplified with high ucibility. The term "reproducibility" used herein refers to an extent of concordance among nucleic acid fragments amplified by a ity of nucleic acid amplification reactions carried out with the use of the same template and the same random . That is, the term "high reproducibility (or the expression "reproducibility is high")" refers to a high extent of concordance among nucleic acid fragments amplified by a plurality of nucleic acid amplification reactions carried out with the use of the same template and the same random primer.

The extent of reproducibility can be evaluated by, for e, conducting a plurality of nucleic acid amplification reactions with the use of the same template and the same random primer, calculating the Spearman's rank correlation coefficient for the data of the nucleotide sequences of the resulting amplified fragments, and ting the extent of reproducibility on the basis of such coefficient. The Spearman's rank cor— relation coefficient is generally represented by the symbol p (rho). When p (rho) is greater than 0.9, for example, the reproducibility of the amplification reaction of interest can be evaluated to be ient.

Random primer A ce constituting a random primer that can be used for producing a DNA library is not particularly limited. For example, a random primer comprising nu— cleotides having 9 to 30 bases can be used. In particular, a random primer may be composed of any tide sequence comprising 9 to 30 bases, a nucleotide type (i.e., a sequence type) is not particularly limited, and a random primer may be composed of 1 or more types of nucleotide sequences, preferably 1 to 10,000 types of nucleotide sequences, more preferably 1 to 1,000 types of nucleotide sequences, further preferably 1 to 100 types of nucleotide sequences, and most preferably 1 to 96 types of nucleotide sequences. With the use of nucleotides (or a group of tides) within the range mentioned above for a random primer, an amplified nucleic acid fragment can be obtained with higher reproducibility. When a random primer comprises a plurality of nucleotide sequences, it is not necessary that all nucleotide sequences comprise the same number of bases (9 to 30 nucleotides). A random primer may comprise a plurality of nucleotide sequences ed of a different number of bases.

When ing a plurality of types of nucleotide sequences for a random primer, % or more, preferably 50% or more, more preferably 70% or more, and further preferably 90% or more of the entire such sequences exhibit 70% or less, ably 60% or less, more preferably 50% or less, and most ably 40% or less identity. By designing a plurality of types of nucleotides for a random primer exhibiting the identity within such range, an amplified fragment can be obtained over the entire genomic DNA of the target organism species. Thus, uniformity of the amplified fragment can be enhanced.

A tide sequence constituting a random primer is preferably designed to have a G—C content of 5% to 95%, more preferably 10% to 90%, further preferably 15% to 80%, and most preferably 20% to 70%. With the use of an aggregate of nucleotides having the G—C content within the aforementioned range as a random , amplified nucleic acid fragments can be obtained with higher ucibility. G—C content is the percentage of guanine and cytosine contained in the whole nucleotide chain.

In particular, a tide sequence used as a random primer is preferably designed to se continuous bases accounting for 80% or less, more preferably 70% or less, further preferably 60% or less, and most ably 50% or less of the full—length sequence. atively, the number of continuous bases in a nucleotide sequence used as a random primer is preferably 8 or less, more preferably 7 or less, further preferably 6 or less, and most preferably 5 or less. With the use of an aggregate of tides comprising the number of continuous bases within the aforementioned range as a random primer, amplified nucleic acid fragments can be obtained with higher repro— ducibility.

In addition, it is preferable that a nucleotide sequence used as a random primer be designed to not comprise a complementary region of 6 or more, more preferably 5 or more, and further ably 4 or more bases in a molecule. Thus, double strand ion ing in a molecule can be prevented, and amplified nucleic acid fragments can be obtained with higher reproducibility.

When a plurality of types of nucleotide sequences are designed as random primers, in particular, it is preferable that a plurality of tide sequences be designed to not comprise complementary regions of 6 or more, more preferably 5 or more, and further preferably 4 or more bases among a plurality of types of nucleotide ces. Thus, double strand formation occurring between nucleotide ces can be prevented, and amplified nucleic acid fragments can be obtained with higher reproducibility.

When a plurality of nucleotide sequences are designed as random primers, in on, it is preferable that such sequences be designed to not comprise com— plementary regions of 6 or more, more preferably 5 or more, and further preferably 5 or more bases at the 3’ us. Thus, double strand formation occurring between nu— cleotide sequences can be prevented, and ied nucleic acid fragments can be obtained with higher reproducibility.

The terms "complementary regions" and "complementary sequences" refer to, for example, regions and sequences exhibiting 80% to 100% identity to each other (e.g., regions and sequences each comprising 5 bases in which 4 or 5 bases are com— plementary to each other) or regions and sequences exhibiting 90% to 100% identity to each other (e.g., regions and sequences each comprising 5 bases in which 5 bases are complementary to each other).

Further, a nucleotide sequence used as a random primer is preferably designed to have a Tm value suitable for thermal cycling conditions (in particular, an annealing temperature) of a nucleic acid amplification reaction. A Tm value can be calculated by a conventional , such as the nearest neighbor base pair approach, the e method, and the GC% , although a method of calculation is not particularly limited thereto. Specifically, a tide sequence used as a random primer is preferably designed to have a Tm value of 10 to 85 degrees C, more preferably 12 to 75 degrees C, further preferably 14 to 70 degrees C, and most preferably 16 to 65 degrees C. By designing a random primer to have a Tm value within the afore— mentioned range, amplified c acid fragments can be obtained with higher repro— ducibility under given l cycling conditions (in particular, at a given annealing temperature) of the nucleic acid amplification reaction.

When a plurality of nucleotide sequences are designed as random primers, in addition, a variation for Tm among a plurality of nucleotide ces is ably 50 degrees C or less, more preferably 45 degrees C or less, further preferably 40 degrees C or less, and most preferably 35 degrees C or less. By designing random primers while adjusting a variation for Tm among a plurality of nucleotide ces within the range mentioned above, amplified nucleic acid fragments can be obtained with higher reproducibility under given thermal cycling conditions (in ular, at a given annealing temperature) of the nucleic acid amplification on.

Nucleic acid ampliﬁcation reaction When producing a DNA y, many DNA fragments are obtained via the c acid amplification reaction carried out with the use of random s and genomic DNA as a template described above. At the time of the nucleic acid amplification reaction, in particular, the concentration of random primes in a reaction on is prescribed higher than the concentration of primers in a conventional nucleic acid am— ation reaction. Thus, many DNA fragments can be obtained with the use of genomic DNA as a template while ing high reproducibility. Such many DNA fragments can be used for a DNA library that can be used for genotyping and other purposes. [003 l] A nucleic acid amplification reaction is aimed at synthesis of amplified fragments in a reaction solution containing genomic DNA as a template, the random primers, DNA polymerase, deoxynucleoside triphosphates as a substrate (i.e., dNTP, which is a mixture of dATP, dCTP, dTTP, and dGTP), and a buffer under the given thermal cycling conditions. It is necessary that a c acid amplification reaction be carried out in a reaction solution containing Mg2+ at a given concentration. In the reaction solution of the composition described above, the buffer contains MgClz. When the buffer does not contain MgClz, the reaction solution of the composition described above further contains MgClz.

In a nucleic acid amplification reaction, in particular, it is preferable that the con— centration of random primers be tely determined in accordance with the base lengths of the random primers. When a plurality of types of tide sequences having different numbers of bases are used as random primers, the number of bases constituting the random primers may be the average of such plurality of nucleotide ces (the e may be a simple average or the weight average taking the amount of nucleotides into account).

Specifically, a nucleic acid amplification reaction is carried out with the use of a random primer sing 9 to 30 bases at a concentration of 4 to 200 microM, and preferably at 4 to 100 microM. Under such conditions, many amplified fragments, and, in particular, many amplified fragments comprising 100 to 500 bases, can be obtained via a nucleic acid ication reaction while achieving high reproducibility.

When a random primer comprises 9 to 10 bases, more specifically, the concentration of such random primer is preferably 40 to 60 microM. When a random primer comprises 10 to 14 bases, it is preferable that the concentration of such random primer satisfy the conditions defined by an inequation: y > 3E + 08x5974 and be 100 microM H H or less, provided that the base length of the random primer is represented by y and the concentration of the random primer is represented by "x." When a random primer comprises 14 to 18 bases, the concentration of such random primer is preferably 4 to 100 microM. When a random primer comprises 18 to 28 bases, it is preferable that the concentration of such random primer be 4 microM or more and satisfy the conditions defined by an inequation: y < 8E + 533. When a random primer comprises 28 to 29 bases, the concentration of such random primer is preferably 6 to 10 microM. By designating the random primer concentration in accordance with the number of bases constituting the random primer as described above, many amplified fragments can be obtained with more certainty while achieving high repro— lity.

As bed in the examples below, the inequations: y > 3E + 08x*6-974 and y < 8E +08x*5-533, are developed to be able to represent the concentration of a random primer at which many DNA fragments comprising 100 to 500 bases can be obtained with high reproducibility as a result of thorough tion of the correlation between random primer length and random primer concentration.

While the amount of genomic DNA serving as a template in a nucleic acid ampli— fication reaction is not particularly d, it is preferably 0.1 to 1000 ng, more preferably 1 to 500 ng, further preferably 5 to 200 ng, and most preferably 10 to 100 ng, when the amount of the on on is 50 microliters. By designating the amount of genomic DNA as a template within such range, many amplified fragments can be obtained without inhibiting the amplification on from a random primer, while achieving high reproducibility.

Genomic DNA can be prepared in accordance with a conventional technique without particular limitation. With the use of a commercialized kit, also, genomic DNA can be easily ed from a target organism species. Genomic DNA extracted from an organism in accordance with a conventional technique or with the use of a commer— cialized kit may be used without r processing, genomic DNA extracted from an organism and then purified may be used, or c DNA subjected to restriction enzyme treatment or ultrasonic treatment may be used.

DNA polymerase used in a nucleic acid amplification reaction is not particularly limited, and an enzyme having DNA rase activity under thermal cycling conditions for a nucleic acid amplification reaction can be used. Specifically, heat— stable DNA polymerase used for a general nucleic acid amplification reaction can be used. Examples of DNA polymerases e thermophilic bacteria—derived DNA polymerase, such as Taq DNA polymerase, and hyperthermophilic archaea—derived DNA polymerase, such as KOD DNA rase and Pfu DNA polymerase. In a nucleic acid amplification on, it is particularly preferable that Pfu DNA polymerase be used as DNA polymerase in combination with the random primer 2017/023343 described above. With the use of such DNA polymerase, many amplified fragments can be obtained with more certainty while achieving high ucibility.

In a nucleic acid amplification reaction, the concentration of ucleoside triphosphate as a substrate (i.e., dNTP, which is a mixture of dATP, dCTP, dTTP, and dGTP) is not particularly limited, and it can be 5 microM to 0.6 mM, preferably 10 microM to 0.4 mM, and more preferably 20 microM to 0.2 mM. By designating the tration of dNTP serving as a substrate within such range, errors caused by incorrect incorporation by DNA rase can be prevented, and many amplified fragments can be obtained while achieving high reproducibility.

A buffer used in a nucleic acid amplification reaction is not particularly limited. For example, a on comprising MgClz as bed above, Tris—HCl (pH 8.3), and KCl can be used. The concentration of Mg2+ is not particularly limited. For example, it can be 0.1 to 4.0 mM, preferably 0.2 to 3.0 mM, more preferably 0.3 to 2.0 mM, and further preferably 0.5 to 1.5 mM. By designating the tration of Mg2+ in the reaction solution within such range, many amplified fragments can be obtained while achieving high reproducibility. [004 l] Thermal cycling conditions of a nucleic acid amplification reaction are not par— ticularly limited, and a common thermal cycle can be adopted. A ic example of a thermal cycle comprises a first step of l denaturation in which genomic DNA as a template is dissociated into single strands, a cycle comprising thermal denaturation, annealing, and ion repeated a plurality of times (e.g., 20 to 40 times), a step of extension for a given period of time according to need, and the final step of storage.

Thermal denaturation can be performed at, for example, 93 to 99 degrees C, preferably 95 to 98 s C, and more preferably 97 to 98 degrees C. Annealing can be performed at, for example, 30 to 70 s C, preferably 35 to 68 degrees C, and more preferably 37 to 65 degrees C, although it varies depending on a Tm value of the random primer. Extension can be performed at, for example, 70 to 76 degrees C, preferably 71 to 75 degrees C, and more preferably 72 to 74 degrees C. Storage can be performed at, for example, 4 degrees C.

The first step of thermal denaturation can be performed within the temperature range bed above for a period of, for example, 5 seconds to 10 minutes, preferably 10 seconds to 5 minutes, and more preferably 30 seconds to 2 minutes. In the cycle comprising "thermal denaturation, annealing, and extension," thermal denaturation can be d out within the temperature range described above for a period of, for example, 2 seconds to 5 minutes, preferably 5 seconds to 2 minutes, and more preferably 10 seconds to 1 minute. In the cycle comprising "thermal ration, annealing, and ion," annealing can be carried out within the temperature range described above for a period of, for example, 1 second to 3 minutes, preferably 3 seconds to 2 minutes, and more preferably 5 seconds to 1 . In the cycle comprising "thermal ration, annealing, and extension," extension can be carried out within the temperature range described above for a period of, for example, 1 second to 3 minutes, ably 3 seconds to 2 minutes, and more preferably 5 seconds to 1 minute.

When producing a DNA library, amplified fragments may be obtained by a nucleic acid amplification on that employs a hot start . The hot start method is intended to prevent mis—priming or non— specific amplification caused by primer—dimer formation prior the cycle comprising "thermal denaturation, annealing, and extension." The hot start method involves the use of an enzyme in which DNA polymerase activity has been suppressed by binding an anti—DNA polymerase dy thereto or chemical modification thereof. Thus, DNA polymerase ty can be suppressed and a non— specific reaction prior to the l cycle can be prevented. According to the hot start method, a temperature is set high in the first thermal cycle, DNA polymerase activity is thus red, and the subsequent nucleic acid amplification reaction is then allowed to proceed.

As bed above, many amplified fragments can be obtained with the use of genomic DNA as a te and a random primer by conducting a nucleic acid ampli— fication reaction with the use of a random primer comprising 9 to 30 bases and pre— scribing the concentration thereof to 4 to 200 microM in a reaction solution. With the use of the random primer comprising 9 to 30 bases by prescribing the concentration thereof to 4 to 200 microM in a reaction on, a nucleic acid amplification reaction can be performed with very high reproducibility. According to the nucleic acid ampli— fication reaction, specifically, many amplified fragments can be obtained while achieving very high reproducibility. Accordingly, such many amplified nts can be used for a DNA library in genetic analysis targeting genomic DNA.

By performing a c acid amplification reaction with the use of the random primer comprising 9 to 30 bases and prescribing the concentration thereof in a reaction solution to 4 to 200 , in particular, many amplified fragments comprising about 100 to 500 bases can be obtained with the use of genomic DNA as a template. Such many ied fragments comprising about 100 to 500 bases are suitable for mass analysis of nucleotide sequences with the use of, for example, a next—generation cer, and highly accurate sequence information can thus be obtained. According to the present invention, accordingly, a DNA library, including DNA fragments comprising about 100 to 500 bases, can be produced.

By performing a nucleic acid amplification reaction with the use of the random primer comprising 9 to 30 bases and prescribing the concentration thereof to 4 to 200 microM in a reaction solution, in particular, the entire genomic DNA can be uniformly amplified. In other words, amplified DNA fragments are not obtained from a particular region of genomic DNA by the nucleic acid amplification reaction with the use of such random primer, but amplified fragments are obtained from the entire . ing to the present invention, specifically, a DNA library can be produced uniformly across the entire genome.

DNA probe In the present invention, the term "DNA probe" refers to a DNA fragment that has a nucleotide sequence complementary to the target DNA fragment and is able to ize to such DNA fragment. A DNA probe that is applicable to a so—called oligonucleotide microarray is particularly preferable. An oligonucleotide microarray is a microarray in which oligonucleotides comprising nucleotide ces of interest are synthesized on a support and the synthesized ucleotides are used as DNA probes. The synthesized oligonucleotides g as DNA probes comprise, for example, 20 to 100 bases, preferably 30 to 90 bases, and more preferably 50 to 60 bases.

The DNA probes designed in accordance with the present invention may be applied to a microarray comprising the synthesized oligonucleotides with the base length described above immobilized on a support, as with the case of the led rd— type microarray. Specifically, the DNA probes designed in accordance with the present invention can be applied to any microarrays according to conventional techniques.

Thus, the DNA probes designed in accordance with the present invention can be applied to a microarray comprising a ﬂat substrate, such as a glass or silicone substrate, as a support and a bead array comprising a microbead support.

According to the method for producing a DNA probe of the present invention, a nu— cleotide sequence of a DNA probe is designed to detect a DNA fragment (a DNA library) on the basis of the nucleotide sequence of the DNA fragment. ically, the nucleotide sequence of the DNA fragment (the DNA library) produced in the manner described above is first determined, and a nucleotide sequence of a DNA probe is designed based on the determined nucleotide sequence. A method for ining a tide sequence of a DNA fragment is not particularly limited. For example, a DNA sequencer in accordance with the Sanger method or a next—generation sequencer can be used. While a next—generation sequencer is not ularly limited, such sequencer is also referred to as a second—generation sequencer, and such sequencer is an apparatus for tide sequencing that is capable of simultaneous determination of tide sequences of l tens of millions of DNA fragments. A sequencing principle of the eneration sequencer is not particularly limited. For example, se— quencing can be carried out in ance with the method in which target DNA is amplified on ﬂow cells and sequencing is carried out while conducting synthesis with the use of bridge PCR method and sequencing—by—synthesis method, or in accordance with emulsion PCR method and the method of Pyrosequencing in which sequencing is carried out by assaying the amount of osphoric acids released at the time of and DNA sis. More specific examples of next—generation sequencers e q, MiSeq, NextSeq, HiSeq, and HiSeq X Series (Illumina) and Roche 454 GS FLX sequencers (Roche). [005 l] Subsequently, a DNA probe is designed to comprise, for example, a nucleotide sequence complementary to the nucleotide sequence of the DNA nt (the DNA library) described above. More specifically, a region or a plurality of s of the base lengths shorter than those of the DNA fragment (the DNA library) and ng at least a part of the DNA fragment (the DNA library) is/are identified, and the identified one or more regions are designed as probes for detecting the DNA fragment (the DNA library).

When a plurality of regions are designed for a particular DNA fragment, such DNA fragment is to be detected with the use of a plurality of DNA probes. A region may be designed for a particular DNA fragment, and two or more regions may be designed for another DNA fragment. Specifically, a different number of regions; that is, DNA probes, may be designed for each DNA fragment. When a plurality of DNA probes are to be designed for a DNA nt, parts of such plurality of DNA probes may overlap with each other, or such plurality of DNA probes may be designed with intervals comprising several bases.

The number of bases tuting a DNA probe to be designed in the manner described above is not particularly limited. Such DNA probe can se 20 to 100 bases, preferably 30 to 90 bases, more preferably 40 to 80 bases, and most preferably 50 to 60 bases.

It is particularly preferable that a plurality of regions be designed, in such a manner that the entire region of a genomic DNA fragment, the tide sequence of which had been determined, would be covered with a plurality of regions. In such a case, a plurality of probes can react with a genomic DNA fragment obtained from genomic DNA derived from a particular organism species via restriction enzyme treatment, and such genomic DNA fragment can be detected with the use of such plurality of probes.

A Tm value of a DNA probe is not particularly limited, and it can be 60 to 95 degrees C, ably 70 to 90 degrees C, more preferably 75 to 85 degrees C, and most preferably 78 to 82 degrees C.

When ing DNA fragments from genomic DNAs with the use of random primers as described above, DNA fragments are obtained from a plurality of different genomic DNAs, and nucleotide sequences of these DNA fragments with different origins can be determined independently from each other. By comparing the de— termined nucleotide sequences, regions having different nucleotide sequences among the genomic DNAs can be identified. According to the method for producing a DNA probe of the present invention, DNA probes can be designed to comprise regions having different nucleotide sequences among the genomic DNAs thus identified.

Specifically, a DNA probe may be designed to comprise a region of a particular genomic DNA that is different from r genomic DNA, and another DNA probe may be designed to comprise a region of the other genomic DNA that is different from the aforementioned ular genomic DNA. With the use of a pair of DNA probes thus designed, a specific type of genomic DNA to be analyzed can be identified.

The nucleotide ce of the DNA fragment amplified from c DNA with the use of a random primer may be compared with a known nucleotide sequence, and a DNA probe may be designed to comprise a region different from such known nu— cleotide sequence. A known nucleotide sequence can be obtained from a variety of conventional databases. While any databases can be used without particular limitation, the DDBJ database provided by the DNA Data Bank of Japan, the EMBL database ed by the European Bioinformatics Institute, the Genbank database provided by the National Center for Biotechnology Information, the KEGG database provided by the Kyoto Encyclopedia of Genes and Genomes, or a combined database comprising such various databases can be adequately used.

Apparatus for DNA analysis The apparatus for DNA analysis according to the t invention comprises the DNA probes designed in the manner bed above immobilized on a support. An apparatus for DNA analysis comprising DNA probes lized on a support is oc— casionally referred to as a "DNA rray." Specifically, the apparatus for DNA analysis according to the t invention is not limited to a so—called DNA chip sing DNA probes immobilized on a support (i.e., a DNA microarray in a narrow sense), and apparatuses composed to be capable of utilization of DNA probes designed in the manner described above on a support are within the scope of the present invention.

For example, a DNA microarray comprising DNA probes designed in the manner described above can be produced in accordance with a conventional technique. A DNA microarray can be ed by, for example, synthesizing an oligonucleotide comprising a nucleotide sequence of the DNA probe ed in the manner described above on a support based on such nucleotide sequence. A method for oligonucleotide synthesis is not particularly limited, and any conventional technique can be employed.

For example, ucleotide synthesis can be performed on a support by pho— tolithography in ation with al synthesis via light application. Alter— natively, an oligonucleotide comprising a linker molecule having a high affinity with a support surface added to its terminus may be separately synthesized on the basis of the nucleotide sequence of the DNA probe ed in the manner described above, and the resulting oligonucleotide may then be immobilized on a support surface at a particular position. A DNA microarray can also be produced by spotting the DNA probe designed in the manner bed above on a support with the use of a pin—type arrayer or a nozzle-type arrayer.

The DNA microarray thus produced (i.e., the apparatus for DNA analysis) comprises a DNA probe comprising a nucleotide sequence complementary to a DNA fragment ied from genomic DNA derived from a particular type of organism with the use of a random primer at a high concentration. Specifically, the DNA microarray thus produced is intended to detect a DNA fragment amplified from genomic DNA with the use of a random primer at a high concentration with the use of a DNA probe. [006 l] A DNA rray may be any of a microarray using a ﬂat substrate made of glass or silicone as a support, a bead array comprising a microbead support, and a three— dimensional microarray comprising a probe immobilized on an inner wall of a hollow fiber.

Method of c DNA analysis With the use of the DNA probe produced in the manner described above, analysis of c DNA, such as genotyping, can be performed. The DNA probe described above is equivalent to the DNA y produced with the use of a random primer at a high concentration. Such DNA library has very high reproducibility, the size of which is suitable for a next—generation sequencer, and it is uniform across the entire genome.

Accordingly, the DNA y can be used as a DNA marker (it is also referred to as a genetic marker or a gene marker). The term "DNA marker" refers to a region in the genome serving as a marker associated with genetic traits. A DNA marker can be used for, for example, breeding comprising a step of selection with the use of genotype identification, linkage maps, gene mapping, or a marker, back ng using a marker, quantitative trait locus mapping, bulked segregant is, variety identification, or discontinuous nce mapping.

Specifically, a DNA marker can be detected with the use of the DNA probe produced in the manner described above, and breeding comprising a step of ion with the use of genotype identification, linkage maps, gene mapping, and a marker, back ng with the use of a marker, quantitative trait locus mapping, bulked segregant analysis, variety identification, or discontinuous imbalance mapping can be carried out.

More specifically, an example of a method for genomic DNA analysis involving the use of the DNA probe comprises bringing the DNA probe produced in the manner described above into contact with a DNA fragment derived from c DNA of the target of analysis. Such DNA fragment may be prepared with the use of the random 2017/023343 primer that was used for producing the DNA library. Alternatively, a pair of primers that ically amplify the DNA marker of interest may be designed on the basis of the nucleotide sequence of interest, and a DNA fragment may be prepared via a nucleic acid amplification on with the use of the pair of designed primers.

Subsequently, hybridization ing between the DNA probe and the DNA fragment is detected in accordance with a conventional technique. For example, a label is added to the amplified DNA fragment, and hybridization of interest can be thus detected on the basis of the label. Any conventional substance may be used as a label.

Examples of labels that can be used include a ﬂuorescent molecule, a pigment molecule, and a radioactive molecule. A labeled nucleotide may be used in the step of DNA fragment amplification.

When a DNA microarray comprising a DNA probe is used, for example, a labeled DNA fragment is brought into contact with the DNA microarray under given conditions, and a DNA probe immobilized on the DNA microarray is allowed to hybridize to a labeled genomic DNA fragment. In this case, a probe hybridizes to a part of the DNA fragment, and it is preferable that ization be carried out under highly stringent conditions, so that hybridization does not occur in the ce of ch of a base, but it occurs only when the bases completely match. Under such highly stringent conditions, a slight change in single nucleotide polymorphism can be detected.

The stringency ions can be adjusted in terms of reaction temperatures and salt concentrations. At a higher temperature, specifically, higher stringency conditions can be achieved. At a lower salt concentration, higher stringency conditions can be achieved. When a probe comprising 50 to 75 bases is used, for example, higher stringency conditions can be achieved by ting hybridization at 40 to 44 s C with 0.21 SDS and 6x SSC.

Hybridization occurring between a DNA probe and a d DNA fragment can be detected based on a label. After the hybridization reaction between the labeled DNA fragment and the DNA probe, specifically, an unreacted DNA fragment or the like is washed, and a label of the DNA fragment that had specifically hybridized to the DNA probe is then observed. When a label is a ﬂuorescent substance, for example, the ﬂu— orescent wavelength is detected. When a label is a pigment molecule, the pigment wavelength is detected. More specifically, an apparatus used for general DNA mi— croarray analysis, such as a ﬂuorescence detector or image er, can be used.

In particular, DNA fragments ied using genomic DNA as a template and a random primer at a high concentration can be ed with the use of such DNA probe. When a DNA probe comprising regions that are ent among a plurality of different genomic DNAs is used, the genomic DNA as the target of analysis can be analyzed in accordance with the DNA probe to which a DNA fragment d from the genomic DNA as the target of analysis had hybridized. For example, a DNA probe ng with a DNA marker comprising differences in tide sequences among genomic DNAs of relative species may be used, so that the species of the genomic DNA as the target of analysis can be fied.

Examples Hereafter, the present invention is described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited to these examples.

Example 1 1. Flow chart In this example, a DNA library was prepared via PCR using genomic DNAs extracted from various types of organism species as templates and various sets of random primers in accordance with the ﬂow chart shown in Fig. 1. With the use of the prepared DNA library, also, sequence analysis was performed with the use of a so— called next—generation sequencer, and the genotype was analyzed based on the read data. 2. Materials In this example, c DNAs were extracted from the sugarcane varieties NiFS and Ni9, 22 hybrid progeny lines thereof, and the rice y Nipponbare using the DNeasy Plant Mini kit (QIAGEN), and the extracted c DNAs were purified.

The purified genomic DNAs were used as NiFS—derived genomic DNA, rived genomic DNA, 22 hybrid sugarcane progeny—derived genomic DNAs, and Nipponbare—derived genomic DNA, respectively. In Example 1, human genomic DNA was purchased from TakaraBio and used as human—derived genomic DNA. 3. Method 3.1 Correlation between PCR ion and DNA fragment size 3.1.1 Random primer ing In order to design random primers, GC content was set between 20% and 70%, and the number of continuous bases was adjusted to 5 or fewer. ce length was set at 16 levels (i.e., 8,9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, 30, and 35). For each sequence length, 96 types of nucleotide sequences were designed, and 96 sets of random s were prepared. Concerning 10—base primers, 6 sets of random primers each comprising 96 types of random primers were designed (these 6 sets are referred to as 10—base primer A to 10—base primer F, respectively). In this example, specifically, 21 different sets of random primers were prepared.

Tables 1 to 21 show nucleotide sequences of random primers contained in such 21 different sets of random primers.

[Table 1-1] Table 1 List of random primers (IO-base primers A) No Primer sequence Z0 Primer ce 1 AGACGTCGTT 0000 GCCGAATAGT 2 GAGGCGATAT 043-00 GTACCTAGGC 3 GTGCGAACGT 35 GCTTACATGA 4 TTATACTGCC 36 TCCACGTAGT CAAGTTCGCA 37 AGAGGCCATC 37 ACAAGGTAGT (A 8 CGGTGATGCT 33 7 ACACAGCGAC 39 CACTGTGCTT 00 LG TTACCGATGT CATGATGGCT O CACAGAGTCG 41 CATG |-‘ GCGT H O 4M CACACACTGT N 11 GTGA ._. I—l 00 CAGAATCATA pl}. 00 12 GTCTGTTCGC p—I [\3 ATCGTCTACG l 00 ACCTGTCCAC ,_. DJ 45 ATAC IIC11,.p. .p. 1 gs. CCGCAATGAC H rh ACAAGCGCAC 907 CTGCCGATCA ,_. (II 47 GCTTAGATGT uh .q l 0'3 TACACGGAGC )—| 0‘) 48 TGCATTCTGG a00 17 CCGCATTCAT I—‘ ~q 49 ACCA #2. L0 18 GACTCTAGAC H CD 50 AGGCACTCGT U1 0 19 GGAGAACTTA 1—: ED 0'! 1 CTGCATGTGA Ip—r TCCGGTATGC [\3 D U1 2 ACCACGCCTA O1 [\3 [\3 1 GGTCAGGAGT [\J ,_. 53 GAGGTCGTAC ()1 DJ 2 ACATTGGCAG [\3 DJ U14 CTGT U1 pk. 3 CGTAGACTGC [\3 OJ 015 TGCCAACTGA an O1 [\D4 AGACTGTACT [\3 kh- 56 ccrcrrccer 01 G: TAGACGCAGT 2 O1 57 GTAGAGAGTT 0‘! --.‘l 26 CCGATAATCT [\3 C73 U1 TACAGCGTAA U1 00 27 GAGAGCTAGT [\3 “-51 U1 TGACGTGATG C11 LO GTACCGCGTT I[\3 AGACGTCGGT GACTTGCGCA Ito 61 CGCTAGGTTC p-r CGTGATTGCG 00 O 62 GCCTTATAGC ATCGTCTCTG ,_. 63 CCTTCGATCT CGTAGCTACG 32 64 AGGCAACGTG 0363mm WM.2.

[Table 1—2] O Primer sequence SEQ ID NO: 65 TGAGCGGTGT 65 GTGTCGAACG m. 67 CGATGTTGCG 7 AACAAGACAC GATGCTGGTT 7O ACCGGTAGTC --..] 71 GTGACTAGCA “J p... 2 AGCCTATATT *4 [\D 73 TCGTGAGCTT 74 ACACTATGGC -q-q rib-DJ 75 GACTCTGTCG .4 01 76 TCGATGATGC ---.l CT) --Zl'-Zl 7 CTTGGACACT -.] .q 8 GGCTGATCGT HS] .q9 ACTCACAGGC II '-~'l {.D ATGTGCGTAC CO 1 CACCATCGAT 00 t—t 82 AGCCATTAAC [\3 83 AATCGACTGT AATACTAGCG CDOO #100 85 TCGTCACTGA ll0‘! CTTA 87 GGTCGGTGAT CD HI 88 CATTAGGCGT CD 00 GAGT TTCCGAATAA 91 TGAGCATCGT A 92 GCCACGTAAC (.0 NI—I GAACTACATG LC! TCGTGAGGAC (D TTAA tD U1 m GCTAAGGACC wo 2013/003727 [Table 2— 1] Table 2 List of random primers (IO—base primers B) No Primer sequence No Primer sequence 1 ATAGCCATTA 33 GGTATAGTAC 2 CAGTAATCAT “ 4 CTAATCCACA 3 ACTCCTTAAT 1. 5 GCACCTTATT 4 ATTA 100 36 ATTGACGGTA ATTATGAGGT 101 37 GACATATGGT 133 102 38 GATAGTCGTA 134 7 TCGC OTTAGGTGAT CATACTACTG 107 139 108 140 109 141 47 TATCGTTGGT 8 CGCTTAAGAT 17 TTGGCCATAT 113 A:9 TTAGAACTGG _145 18 TATTACGAGG O GTCATAACGT 146 19 TTATGATCGC U1 1 AGAGCAGTAT 147 AACTTAGGAG 52 CAACATCACT 148 21 TCACAATCGT U1 3 CAGAAGCTTA 149 2 GAGTATATGG 54 AACTAACGTG 150 3 ATCAGGACAA 55 TTATACCGCT 151 N4 GTACTGATAG 120 CH 6 GAATTCGAGA 152 [\3 5 CTTATACTCG 121 7 AACC 153 [\3 6 TAACGGACTA 122 8 TTAA 154 27 GCGTTGTATA 123 59 GCACCTAATT 155 28 CTTAAGTGCT m_155 9 ATACGACTGT ACTGTTATCG [Table 2—2] No Primer sequence 65 AGTATCCTGG 161 66 GGTTGTACAG 162 67 ACCA 163 TGTCGAGCAA 164 GTCGTGTTAC 165 70 GTGCAATAGG 166 71 ACTCGATGCT 167 GAATCGCGTA 168 CGGTCATTGT 169 ATCAGGCGAT 170 GTAAGATGCG 171 6 GGTCTCTTGA 172 7 CTAA 173 8 CTGCGTGATA 174 79 CATACTCGTC 175 ATCTGAGCTC 176 81 ACGGATAGTG 177 CO [\3 ACTGCAATGC 178 3 CGTG 179 4 TAGACTGTCG 180 OO 5 CAGCACTTCA 181 AACATTCGCC 182 87 ACTAGTGCGT 183 88 ACGCTGTTCT L 184 CGTCGAATGC 185 CTCTGACGGT 186 91 GTCGCCATGT 187 (Q GGTCCACGTT 188 93 CGAGCGACTT 189 94 TTGACGCGTG 190 CTGAGAGCCT CGCGCTAACT 192 [Table 3— 1] Table 3 List of random primers (IO-base primers C) SE ID Primer sequence 30 No Primer sequence 1 GGTCGTCAAG 193 33 H040004400 225 2 AGGTTGACCA 194 4 AACTGCAGT 226 3 195 5—_227 4 196 36—_228 197 37 0100041400 229 198 38 1440011000 230 7—1401100040 199 C439 1144 231 10404 200 ,2.0 4044040000 232 0100410440 201 41 0041140401 2 00 DJ 0004010114 2 0101010404 [\3 DJ4 11 1400404010 203 3 0004110040 235 1—- N) 4040040404 204 14>-4 1011000400 230 )— 3 0000 205 45 1400010100 237 14 0011401004 205 0014 238 0441400144 207 7 0000400411 239 16 4040110000 208 48 4140040400 240 17 0400001011 209 49 0040010414 241 18 CGTGAGAGGT 210 0 4400001100 242 19 4410001040 IIU11 0004000114 243 4140014000 212 52 1404040001 244 21 4401041100 213 0004011040 N2 0104000140 ll4 1404400104 245 23 0100041100 U‘l 5 0004110040 247 24 0000400414 215 0004010011 248 0040440144 217 0140001144 249 6 0144004000 218 4414001010 250 N 7 0010040041 219 59 4001 251 28 0400000114 220 0010441000 252 9 4010010400 221 61 4104400000 253 0400040104 62 0000440014 254 31 1010400104 [‘0 [\3 00 63 4044040000 255 32 1404104001 64 0000414010 255 [Table 3—2] No Primer sequence 65 CTTATATGTG GGTCTCATCG CCACCATGTC m ACGAATGTGT m GGTAGTAACA 261 TAAT 262 ATATTGCGCC 263 GACCAATAGT 264 73 AACAACACGG 265 4 ATAGCCGATG 266 75 CGAGAGCATA 267 6 CGAGACATGA 268 -\] 7 CGCCAAGTTA 269 8 TTATAATCGC 270 79 TAGAAGTGCA 271 ATGT 272 81 GCCACTTCGA 273 2 TCCACGGTAC 274 3 CAACTATGCA 275 CAAGGAGGAC 276 GAGGTACCTA 277 GAGCGCATAA 276 ‘---J TCGTCACGTG M .4 L0 88 AACTGTGACA 280 TGAG 281 ACACTGCTCT 91 TACGGTGAGC 92 CGGACTAAGT 284 H3 AAGCCACGTT [\3 00 (TI CAATTACTCG 286 TCTGGCCATA 287 m TCAGGCTAGT [Table 4— 1] Table 4 List of random primers (IO-base s D) . SEQ ID No Prlmer sequence _ No Pruner sequence l TTGACCCGGA 33 CAAGTCAGGA 2 TTTTTATGGT GGGTCGCAAT 3 ATGTGGTGCG 291 CAGCAACCTA 4 AAGGCGCTAG 292 TTCCCGCCAC TCCAACTTTG 293 TGTGCATTTT CCATCCCATC 294 CD 00 ATCAACGACG 7 CAATACGAGG 295 DJ 9 GTGACGTCCA TACC 29 0 CGATCTAGTC GCCTCCTGTA II297 H TTACATCCTG CGAAGGTTGC 298 42 AGCCTTCAAT ll GAGGTGCTAT 299 3 TCCATCCGAT 331 1 N TAGGATAATT 300 ,p.4 GTCT 332 l3 CGTTGTCCTC 14‘:-5 TTCGGTGGAG 333 l4 TGAGACCAGC 6 GACCAGCACA 334 1 U1 TGCCCAAGCT 7 CGGA 335 16 TACTGAATCG 304 TTTTTCTTGA 17 TTACATAGTC 305 49 CATTGCACTG 337 1 00 ACAAAGGAAA 306 50 TGCGGCGATC 333 19 CTCGCTTGGG 307 l ATATTGCGGT 339 CCTTGCGTCA 303 U12 GACGTCGCTC 340 21 TAATTCCGAA 309 53 TCGCTTATCG 341 22 GTGAGCTTGA 4 GCGCAGACAC 3 ATGCCGATTC 311 5 CATGTATTGT 4 GCTTGGGCTT 312 CD ACCT 344 ACAAAGCGCC 313 57 GTGGAGACAA 345 -—6GAAAGCTCTA 314 -—8 CGAAGATTAT 346 -—7TACCGACCGT 315 .—59 TAGCAACTGC 347 319 CACGCCTTAC 320 AGTTGGTTCC [Table 4—2] O Primer sequence 65 TCTTATCAGG CGAGAAGTTC 67 GTGGTAGAAT 355 TAGGCTTGTG 356 TACG 357 7O ACTACCGAGG 358 71 GGTG 359 72 GGACGATCAA 360 .4 3 AACAGTATGC 361 74 TTGGCTGATC 362 75 AGGATTGGAA 363 6 CATATGGAGA 364 7 CTGCAGGTTT 365 8 CTCTCTTTTT 366 79 AGTAGGGGTC 367 ACACCGCAAG 368 81 GAAGCGGGAG 369 2 GATACGGACT 370 3 TACGACGTGT 371 84 GTGCCTCCTT 372 GGTGACTGAT 373 86 ATATCTTACG 374 -.q AATCATACGG 375 88 CTCTTGGGAC 376 GACGACAAAT 377 GTTGCGAGGT 378 91 AAACCGCACC 379 92 GCTAACACGT 380 93 ATCATGAGGG 381 CGTA 382 TCTCGAAAAG 383 m CTCGTAACCA 384 wo 2018/003727 [Table 5— 1] Table 5 List of random s (1 0—base primers E) % C3 No Primer sequence No Primer sequence E:0’2 GTTACACACG (.0 3 )—]TCCGGTTAT 2 CGTGAAGGGT 386 4 ATAAACTGT 3 ACGAGCATCT 5 ACAGTTGCC 4 ACGAGGGATT 388 6 GATGGCGAA TCGG 389 DJ 7 CGACGTCAG CACGGCTAGG 390 -(2) ATGGTGCAA 7 CGTGACTCTC 391 {D C)ACGACAGTC 423 ﬂ CGCA 392 GTCACCGTCC 424 u—393 425 396 428 397 429 398 430 399 431 402 434 403 435 404 436 405 53 ACGCAATAAG 437 406 54 AAGGTGCATC 438 CGCGTAGATA 439 NI-P- GCGAGGATCG U16 CGAGCAGTGC CACGTTTACT 409 (II 7 ATACGTGACG N01 TACCACCACG vb |—| O 58 AGATTGCGCG 442 TTAACAGGAC U‘l 9 ACGTGATGCC 443 [\3 DD GCTGTATAAC 412 GTACGCATCG 444 GTTGCTGGCA 413 61 TCCCGACTTA GCCA 414 62 GTTTTTACAC _ CO 1 CTGCGGTTGT 1-P- p—I U‘l 63 ('1CTGAGCGTG 32 TAGATCAGCG 64 C“)GGCATTGTA [Table 5—2] No Primer sequence 65 GCGT ATGGCCAGAC 67 CTTAGCATGC ACAACACCTG TATC 70 CATGCTACAC --l--] '1 AAAGCGGGCG 2 AGATCGCCGT 456 73 TATT 457 “HI4 AATGGCAGAC 458 —.J5 GTATAACGTG 459 .4 6 ATGTGCGTCA 460 '5] q CCTGCCAACT 461 78 TTTATAACTC 462 79 ACGGTTACGC 463 TAGCCTCTTG 00 1 TCGCGAAGTT 465 82 GTCTACAACC 466 83 GTCTACTGCG 467 4 GTTGCGTCTC 468 85 GGGCCGCTAA 469 GTACGTCGGA 470 87 AGCGAGAGAC 471 88 TGGCTACGGT 472 AGGCATCACG 473 TAGCTCCTCG 474 91 GGCTAGTCAG 475 (D2 CTCACTTTAT 476 93 ACGGCCACGT 477 AGCGTATATC 478 GACACGTCTA 479 m GCCAGCGTAC W0 2018/003727 [Table 6— 1] Table 6 List of random primers (IO-base primers F) . SEQ ID aner sequence, 481 818 488 81881848111 n_484 GTCTAGTTGC 516 485 817 II— 488 818 487 818 488 528 TGGCGTGAGA ,1: 1 CATT 521 TTGCCAGGCT 490 2 AGGTCCTCGT 522 11 GTTATACACA 491 3 TTGTGCCTGC 523 12 AGTGCCAACT 492 44 ACCGCCTGTA 524 H 3 TCACGTAGCA 493 5 CAGG 525 14 TAATTCAGCG 494 0'3 GCACACAACT 526 1 AAGTATCGTC 495 7 TGAGCACTTA 527 16 CACAGTTACT 496 8 GTGCCGCATA 528 17 CCTTACCGTG 497 9 TCGC 529 18 TCGT 498 50 ACACTTAGGT 530 19 CGCGTAAGAC 499 U‘l 1 CGTGCCGTGA 531 TTCGCACCAG 500 U1 t0 TTACTAATCA 532 H._. CACGAACAGA 501 3 GTGGCAGGTA 533 GTTGGACATT 502 4 GCGCGATATG 534 GGTGCTTAAG 503 55 CGTT 535 24 TCGGTCTCGT 504 U1 6 ATCAGGAGTG 536 TCTAGTACGC 505 7 GCCAGTAAGT 537 6 TTAGGCCGAG 506 U1 8 GCAAGAAGCA 538 27 CGTCAAGAGC 507 59 AACTCCGCCA 539 28 ACATGTCTAC 508 ACTTGAGCCT 540 9 ATCGTTACGT 61 CGTGATCGTG ACGGATCGTT (II >—- O 62 AATTAGCGAA 31 AATCTTGGCG 63 ACTTCCTTAG 32 AGTATCTGGT 64 TGTGCTGATA 544 [Table 6—2] 7’ o Primer sequence SEQUDNO 65 AGGCGGCTGA 545 67 ACGCGTCTAA 547 m GCGAATGTAC 548 E 5515515555 549 5555555515 550 5555115555 551 5551155551 552 73 5155555515 553 4 5515555555 554 5515 555 75 5151555511 555 77 5515555155 557 78 5515151515 555 79 5551151555 559 5155155155 550 81 1555 551 C132 5555115155 552 3 5555511555 553 84 5551555551 554 85 5511515555 555 5155555551 555 87 5115555155 557 88 5551 568 5511515551 559 5515551555 570 91 5555551155 571 {D 2 5555115515 572 93 5155155151 U'I --.] 3 4 5155515551 574 95 1555515515 575 5555555151 575 W0 2018/003727 [Table 7— 1] Table 7 List of random primers (8-base primers) No Primer sequence No ’11rimer sequence 1 CTATCTTG ll20577 33 CGTCAAGT 2 GT 578 34 AAGTAGAC 3 ACATGCGA 579 35 TCAGACAA 4 ACCAATGG 580 36 TCCTTGAC TGCGTTGA 581 37 GTAGCTGT GACATGTC 582 8 CGTCGTAA 7 57 583 OJ 9 CCAATGGA ACATCGCA 584 0 TTGAGAGA GAAGACGA I35 41 CC TCGATAGA 555 [\J TCTAGTAC 11 TCTTGCAA 587 3 GAGGAAGT 1 AGCAAGTT CI] 00 CO 4 GCGTATTG 13 TTCATGGA 589 ,7;5 AAGTAGCT 621 14 CG 590 TGAACCTT 622 CGGTATGT _ 7 TGTGTTAC 1 6': ACCACTAC 48 TAACCTGA 624 17 TCGCTTAT 593 GCTATTCC 525 18 TCTCGACT 594 577715575 525 19 GAATCGGT 595 CAGGATAA 527 0 GTTACAAG 595 ACCGTAGT 528 21 57575755 597 53 CCGTGTAT 629 [\32 TGGTAGAA 54 TCCACTCT 630 N3 ATACTGCG 599 CF] 0'] TAGCTCAT 631 24 AACTCGTC 550 CGCTAATA 532 MN5 ATATGTGC 501 TACCTCTG 533 0'} 55577555 502 TGCACTAC 534 27 GATCATGT 503 CTTGGAAG 535 28 77577557 m AATGCACG 535 29 CCTCTTAG CACTGTTA 537 03 0 TCACAGCT “ G3 [\3 TCGACTAG G: 03 DO DJ 1 AC CTAGGTTA 639 32 AT GCAGATGT 640 [Table 7—2] No Primer sequence 65 AGTTCAGA l2641 61664164 642 67 16611466 643 46614664 644 64 645 70 66164641 646 71 16641641 647 2 41416646 01 6h 00 —..] 3 11616646 4 11464664 650 75 14461466 651 .4 O3 61416466 652 a} 7 61161646 653 8 CGTTCTCT 654 79 61646141 655 TCGTTAGC 656 81 41661614 657 [\3 64646644 658 83 46466644 659 4 16646114 85 44166646 661 41646616 662 87 ACTGTGCA 663 88 64 664 66416644 665 46646141 91 66446641 92 CCTTGTGT CEO} 0101 00-5] 93 16666414 m 4 46644166 670 95 41661446 671 64416161 672 wo 2018/003727 [Table 8— 1] Table 8 List of random primers (9-base primers) _ SEQ ID .

E—678 II_ 680 II— 681 684 716 685 717 686 46 GATAAGCGT 718 687 47 ATATCTGCG 719 48 ACTTAGACG 720 1'7_TATGACACT ,4;9 ATCACCGTA 721 1 DO—ATTAACGCT m O TAAGACACG ---CI [\3 N 19 TAGGACAAT 691 1 AATGCCGTA 723 AAGACGTTA 692 52 AATCACGTG 724 21 TATAAGCGT 693 53 TCGTTAGTC 725 2 GGC 694 4 CATCATGTC 726 3 CTCGAGATC 695 U‘IU'I 5 GGT 4 ATGGTGAGG m 016 TGCATAGTG [\3 5 ATGTCGACG 697 GAGCGTTAT - 729 26 GACGTCTGA 698 U"! oo ACA -\] DJ C N 7 TACACTGCG m TTCGCGTTA 28 ATCGTCAGG 700 m GTGTTAACG 9 TGCACGTAC GACACTGAA 0 GTCGTGCAT CTGTTATCG 31 GAGTGTTAC ‘---JOW I0') C)GTCGTTAT 32 AGACTGTAC 64 ('JGAGAGTAT WO 03727 [Table 8—2] No Primer sequence 65 ATACAGTCC 737 AATTCACGC 738 67 TATGTGCAC 739 GATGACGTA 740 GATGCGATA . 741 70 GAGCGATTA 742 71 TGTCACAGA 743 72 CCG 744 q3 CATAACGAG 745 4 CGTATACCT 746 75 TATCACGTG 747 --J 6 GAACGTTAC 748 7 GTCGTATAC 749 78 ATGTCGACA 750 79 ATACAGCAC 751 TACTTACGC 752 81 AACTACGGT 753 82 TAGAACGGT 754 83 GAATGTCAC 755 TGTACGTCT 756 85 GCG 757 TTGAACGCT 758 87 AATCAGGAC 759 88 ATTCGCACA 760 CCATGTACT 761 TGTCCTGTT 762 91 TAATTGCGC 763 92 GATAGTGTG .q O) H: 93 ATAGACGCA 765 94 TGTACCGTT 766 ATTGTCGCA 767 m GTCACGTAA [Table 9— 1] Table 9 List of random primers (1 1-base primers) H_774 II— 776 2 79977909769 - 11 AACTT 779 3 GTAGAGGTTGG 12 TCAGATGTCCG 780 4 CTCTTGCCTCA 812 13 CTGCTTATCGT 781 11:. 5 ATCGTGAAGTG 813 14 ACATTCGCACA 782 .12.6 ACTAT 814 CCTTAATGCAT 7 OO (.0 7 CACCAGAATGT 815 16 GGCTAGCTACT 784 ﬁ00 GAGTGAACAAC 816 17 TTCCAGTTGGC 785 49 TAACGTTACGC 817 18 GAGTCACAAGG 786 0 CTTGGATCTTG 19 CAGAAGGTTCA 787 1 GTTCCAACGTT 819 TCAACGTGCAG 788 U‘t2 CAAGGACCGTA 820 21 CAAGCTTACTA 789 53 GACTTCACGCA 821 '2 AGAACTCGTTG 790 4 CACACTACTGG 822 3 CCGATACAGAG 791 5 TCAGATGAATC 823 4 GTACGCTGATC 792 U‘l 6 TATGGATCTGG 824 TCCTCAGTGAA 793 57 TCTTAGGTGTG 825 26 GAGCCAACATT 794 8 TGTCAGCGTCA 826 7 GAGATCGATGG 795 59 GTCTAGGACAG 827 28 ATCGTCAGCTG 796 GCCTCTTCATA 828 9 GAAGCACACGT 797 61 AGAAGTGTTAC 829 0 ATCACGCAACC 798 62 CATGAGGCTTG 830 31 TCGAATAGTCG 799 63 TGGATTGCTCA 831 32 CGTCT 800 64 ATCTACCTAAG 832 [Table 9—2] No Primer sequence 65 ATGAGCAGTGA 66 CCAGGAGATAC 67 CCGTTATACTT CTCAGTACAAG GGTGATCGTAG 70 CGAACGAGACA 71 ACTACGAGCTT 839 2 TTGCCACAGCA 840 3 GTCAACTCTAC 74 GTGTC 842 GGAATGGACTT 843 6 CGAGAACATAA 844 77 ACCTGGTCAGT 845 78 CGAACGACACA 846 79 AGTCTAGCCAT 847 AGATG 848 1 GGTGCGTTAGT 849 82 ATTGTGTCCGA 850 83 GCAGACATTAA 851 ATTGGCTCATG 852 85 GAGGTTACATG 853 CCTATAGGACC 00 7 TTAGACGGTCT 855 88 GATTGACGCAC 856 AAGACACCTCG 857 TCGAATAATCG 858 91 TCTATGTCGGA 859 2 TCGCATGAACC 860 93 TGTTATGTCTC 861 94 CTACA 95 ATCGTTCAGCC TACCGCAAGCA [Table 10—1] Table 10 List of random primers (IZ-base primers) No Primer sequence No Primer sequence NO NO 1 GCTGTTGAACCG 33 ACTGAGGCGTTC m(D7 2 ATACTCCGAGAT 34 TAAGGCTGACAT 898 3 CTTAAGGAGCGC 867 35 AGTTCGCATACA 899 4 TATACTACAAGC 868 6 GCAGAATTGCGA TAGTGGTCGTCA 869 (.000 7 GAAGAA 901 GTGCTTCAGGAG 870 8 AGAAGTCGCCTC 902 7 GACGCATACCTC 871 9 TTCGCGTTATTG 903 CCTACCTGTGGA 872 40 TACCTGGTCGGT 904 GCGGTCACATAT 873 q; 1 CGAGGA 905 CTGCATTCACGA 874 42 ACACACTTCTAG 11 CTTCAT 875 3 GGAAGTGATTAA 907 l [\3 TTGTGCTGGACT 876 4 TCCATCAGATAA 908 l3 ATTGAGAGCTAT 877 5 TGTCTGTATCAT 14 TCGCTAATGTAG 878 6 AATTGGCTATAG 910 CTACTGGCACAA 879 7 ACGTCGGAAGGT {D ;_. ‘—A 16 AGAGCCAGTCGT 880 1-58 AGGCATCCGTTG 9 ACCGTCGCTTGA y—ip—n 2 7 AATACTGGCTAA 881 49 9 3 'H191 CTGCATGCATAA 882 50 TACCGTCAAGTG 914 TTGTCACAACTC 883 51 CTCGATATAGTT 915 TGCTAACTCTCC 884 2 CG'I‘CAACGTGGT 916 21 TCTCTAGTTCGG 885 3 TAGTCAACGTAG 917 TTACGTCCGCAA 886 54 TGAGTAGGTCAG 918 3 GTGTTGCTACCA 887 0'! 5 CTTGGCATGTAC 919 4 CGCATGTATGCC 888 01 6 TGCCGAGACTTC 920 CTGATT 889 57 CTAAGACTTAAG 921 6 TAAGATGCTTGA 890 CO TTCTCGTGTGCG 922 27 ATATATCTCAGC 891 9 CACCTGCACGAT 923 8 TTCCTCGTGGTT 892 ATTAAGCCTAAG 924 9 ATCTAG 893 01 1 ACCATG 925 I'm0 CATCCACTAATC 894 62 ACTAACGCGACT 926 31 GCCTCTGGTAAC 895 63 TGCTAT 927 32 AGTCAAGAGATT 896 64 ACGCTGTTAGCA 928 [Table 10—2] E0 Primer ce GTCAACGCTAAG m AGCTTAGGTATG CGCAGGACGATT m AACCGGCTGTCT CACGTG 70 GAATCTTCCGCG 71 AGAGCGTACACG “~31"!2 AAGGCTAATGTC 3 TCTATGTAGACG 4 TCTAGT 75 TTGGTCACACGC 76 GTCGATATATGG «1-»: 7 AACATGGATACG 8 TTCGCAGTTCCT 79 CGCATGTTGTGC 943 TGTTAAGTTGGA 81 CAAGTGTGATGA 945 82 CTGGTACCACGT 946 3 CGCTAGGATCAC 947 4 TGCTCATTACGG —f9 #2.m TGCTCAGTAACA ACGATCATAGCC 950 87 ACGATACGTGGA 951 88 GTTCGATGATGG 952 AAGAGCTGTGCC 953 GGTTGGATCAAC 954 91 GCGCGCTTATGA 955 92 CGTCGATCATCA 956 (D 3 GAGACTGCACTC 957 4 GATAGATCGCAT 958 95 GGCCATCATCAG 959 GGTGTTCCACTG [Table 11— 1] Table 11 List of random primers (14-base primers) NO NO 964 -—m 7 ACCGGCATAAGAAG 967 -—m GGATGCTTCGATAA 968 0 CTCCAGTAATACTA GTGTACCTGAATGT 4:. 1 TGATGCCGATGTGG CGCGGATACACAGA 970 .4;2 GTCATACCGCTTAA 11 TTCCACGGCACTGT 971 .4;3 ACGTTCTCTTGAGA 12 TAGCCAGGCAACAA 972 4 CAGCCATATCGTGT l3 AACACGTA 973 5 TTGAACGTAGCAAT l4 TAACGCTACTCGCG 974 6 ACAATCGCGGTAAT 1 0‘] TAGATAGACGATCT 975 7 GTTCCTGTAGATCC 16 ACTCTTGCAATGCT 976 p52.8 AGAGCCTTACGGCA 17 ACTCGGTTAGGTCG 977 49 AATATGGCGCCACC 1 CATTATCTACGCAT 978 0 ACCATATAGGTTCG 19 CACACCGGCGATTA 979 1 ATGCACCACAGCTG N0 TACGCAGTACTGTG 980 2 CTACTATTGAACAG 21 CAAGCGCGTGAATG 981 U101 3 TGCCATCACTCTAG 22 GAATGGACTGACGA 982 01 A; GCGAACGAGAATCG 3 CTGAAGTT 983 5 GAATCAAGGAGACC 4 TGCGGCAGACCAAT 984 U1 CAACATCTATGCAG AAGGCATAGAGATT 985 0'! 7 CAATCCGTCATGGA 26 TTCTCCTCGCCATG 986 O'I8 AGCTCTTAGCCATA [\D 7 TCATTGGTCGTGAA 987 59 AACAAGGCAACTGG 28 CTATACGA 988 GCTCCTAT 29 ATGATCCTCCACGG 989 61 GTCATCATTAGATG (.0 0 CGTCGTTAGTAATC 2 GCACTAAGTAGCAG 31 TGCACATAGTCTCA 991 0'30: 3 ACCTTACCGGACCT 32 GTCAAGGAGTCACG 992 64 GCTCAGGTATGTCA [Table 11—2] Z0 Primer sequence SEQHDNO 65 TGTCACGAGTTAGT 1025 CAGATGACTTACGT 1026 67 GCGATTGA 1027 GCAGGCAATCTGTA 1028 ACAACAAG 1029 70 CCTTAGATTGATTG 1030 71 AGCCACGAGTGATA 1031 —q [\3 CGATGACTCGTGAC 1032 --..1 3 CTTCGTTCGCCATT 1033 «a4 TCTTGCGTATTGAT 1034 «a 5 CTTAACGTGGTGGC 1035 -..1 6 TGCTGTTACGGAAG 1036 77 CTGAATTAGTTCTC 1037 .48 CCTCCAAGTACAGA 1038 79 CTGGTAATTCGCGG 1039 CGACTGCAATCTGG 1040 81 TGGATCGCGATTGG 1041 82 CGACTATTCCTGCG 1042 3 CAAGTAGGTCCGTC 1043 84 CAGTGTTC 1044 OD 5 TTATTCTCACTACG 1045 CATGTCTTCTTCGT 1046 87 AGGCACATACCATC 1047 88 AGGTTAGAGGATGT 1048 CAACTGGCAAGTGC 1049 CGCTCACATAGAGG 1050 91 GCAATGTCGAGATC 1051 GTTCTGTGGTGCTC 1052 AAGTGATCAGACTA 1053 ATTGAAGGATTCCA 1054 I{D ACGCCATGCTACTA 1055 CTGAAGATGTCTGC 1056 WO 03727 [Table 12—1] Table 12 List of random primers (lG-base s) SEQ ID No Primer sequence 2O Primer sequence NO NO GACAATCTCTGCCGAT 33 AATGACGTTGAAGCCT 2 GGTCCGCCTAATGTAA 34 TCGATTCTATAGGAGT 3 AGCCACAGGCAATTCC 5 CGATAGGTTCAGCTAT 4 AGTTCTCAAC 0300 6 CCATGTTGATAGAATA TGTAACGCATACGACG -.1 GAGCCACTTCTACAGG TATCTCGAATACCAGC 8 GCGAACTCTCGGTAAT 7 ACCGCAACACAGGCAA 39 GACCTGAGTAGCTGGT GGCCAGTAACATGACT 4O CGAGTCTATTAGCCTG GTGAACAGTTAAGGTG 1 GTAGTGCCATACACCT CCAGGATCCGTATTGC hE-nh2 CCAGTGGTCTATAGCA GACCTAGCACTAGACC -pp.3 GTCAGTGCGTTATTGC CCTATTCACG AGTGTCGGAGTGACGA 3 AAGTGCAGTAATGGAA CJ‘I AATCTCCGCTATAGTT 14 TCAACGCGTTCGTCTA 0') CGAGTAGGTCTGACTT 1 AGCGGCCACTATCTAA H3 CTGTCGCTCTAATAAC 1 EU] CTCGGCGCCATATAGA 8 GCTGTCAATATAACTG 17 CGATAACTTAGAAGAA A:9 AGCTCAAGTTGAATCC 1 00 CATAGGATGTGACGCC 50 AATTCATGCTCCTAAC 19 GGCTTGTCGTCGTATC 1 CCAAGGTCTGGTGATA TGAATATTAG 52 CTCCACGTATCTTGAA 1 ACAGTTCGAGTGTCGG 53 TAGCCGAACAACACTT 22 CTCTAACCTGTGACGT 54 AGTACACGACATATGC 3 CGCGCTAATTCAACAA 55 ACGTTCTAGACTCCTG 24 ACTCACGAATGCGGCA U1 G'J CGACTCAAGCACTGCT [\‘JMM “-4015 AATCTTCGGCATTCAT U1 7 TGAAGCTCACGATTAA AAGTATCAGGATCGCG 6'! TATCTAACGTATGGTA AGTAACTCTGCAGACA U1 9 TATACCATGTTCCTTG 28 GGATTGAACATTGTGC TTCCTACGATGACTTC GTGATGCTCACGCATC 61 AATATGTGCC CGTAGCGTAACGGATA 62 GAGTAGAGTCTTGCCA TGCGATGCACCGTTAG 63 GCGAGATGTGGTCCTA CCAGTATGCTCTCAGG 64 AAGCTACACGGACCAC [Table 12—2] 7—4 Primer sequence 65 ATACAACTGGCAACCG CGGTAGATGCTATGCT 67 CCGGTCATCA AGATCGTGCATGCGAT TCCTCGAGACAGCCTT 7O TAGCCGGTACCACTTA 1 GTAAGGCAGCGTGCAA 2 TAGTCTGCTCCTGGTC 73 TGGATTATAGCAGCAG 4 AAGAATGATCAGACAT 75 CAGCGCTATATACCTC a] 6 GAGTAGTACCTCCACC 77 GACGTGATCCTCTAGA 8 GTTCCGTTCACTACGA 79 TGCAAGCACCAGGATG TTAGTTGGCGGCTGAG 81 CAGATGCAGACATACG 82 GACGCTTGATGATTAT 3 TGGATCACGACTAGGA 84 CTCGTCGGTATAACGC 005 AAGCACGGATGCGATT 86 AGATCTTCCGGTGAAC 0300 T GGACAATAGCAACCTG 8 GATAATCGGTTCCAAT CTCAAGCTACAGTTGT GTTGGCATGATGTAGA 91 CAGCATGAGGTAAGTG 92 GCCTCATCACACGTCA (D 3 CTACACATCG 4 TACACGAGGCTTGATC 95 TTCTCGTGTCCGCATT GGTGAAGCAACAGCAT [Table 13—1] Table 13 List of random primers (18-base primersV SEQ ID SEQ ID No Primer sequence 20 Primer sequence I:C) 1 CGAACCGACTGTACAGTT 1153 33 ATGTTCAGTCACAAGCGA 2 CCGACTGCGGATAAGTTA 1154 34 TAGGAAGTGTGTAATAGC 3 CGACAGGTAGGTAAGCAG 1155 DJ 5 AATCCATGTAGCTGTACG 4 TGATACGTTGGTATACAG 1156 36 TCACTGGCATAG CTACTATAGAATACGTAG 1157 DO 7 TTGTCTCTACGTAATATC AGACTGTGGCAATGGCAT 1158 38 GTGGTGCTTGTGACAATT 7 GGAAGACTGATACAACGA 1159 39 CAGCCTACTTGGCTGAGA TATGCACATATAGCGCTT 1160 ATGCATCTGTGT CATGGTAATCGACCGAGG 1161 41 TGTAGAGAGACGAATATA GTCATTGCCGTCATTGCC 1162 42 GCCTACAACCATCCTACT 11 CCTAAGAACTCCGAAGCT 1163 43 GCGTGGCATTGAGATTCA 12 ACCGTACTAGGA 1164 44 GCATGCCAGCTAACTGAG 13 TATTACTGTCACAGCAGG 1165 45 GCGAGTAATCCGGTTGGA ,_1 4 TGAGACAGGCTACGAGTC 1166 GCCTCTACCAGAACGTCA AAGCTATGCGAACACGTT 1167 GTCAGCAGAAGACTGACC 16 GGAGTGAGCCAA 1168 GATAACAGACGTAGCAGG 17 CCACTATGGACATCATGG 1169 CAGGAGATCGCATGTCGT 18 GTGGATAGCTCG 1170 50 CTGGAAGGAATGGAGCCA 19 TCACCGGTTACACATCGC 1171 U1 1 ATTGGTTCTCTACCACAA .AAGATACTGAGATATGGA 1172 0'!2 CTCATTGTTGACGGCTCA 21 GACCTGTTCTTGAACTAG 1173 U1 3 TTCAGGACTGTAGTTCAT II2 AAGTAGAGCTCTCGGTTA 1174 54 AGACCGCACTAACTCAAG 23 CTATGTTCTTACTCTCTT 1175 01 TTGTGCAGACCG CAAGGCTATAAGCGGTTA 1176 CCTATTACTAATAGCTCA 1177 010101 0') GAAGCTAATTAACCGATA 7 ATGGCATGAGTACTTCGG TTCACGTCTGCCAAGCAC 1178 58 GACACGTATGCGTCTAGC ATCGTATAGATCGAGACA 1179 (II 9 GAAGGTACGGAATCTGTT GTCACAGATTCACATCAT 1180 TATAACGTCCGACACTGT GTGCCTGTGAACTATCAG 1181 61 ACATTACCGCCG CAGCGTACAAGATAGTCG 1182 62 GAAGCCAACACTCCTGAC GCATGGCATGGTAGACCT 63 CGAATAACGAGCTGTGAT 32 GGTATGCTACTCTTCGCA 64 GCCTACCGATCGCACTTA WO 03727 [Table 13—2] GO 1 LO 3 [Table 14—1] Table 14 List of random primers (20-base primers) SEQ ID SE ID Primer sequence 0 NO NO ACTGGTAGTAACGTCCACCT AGACTGGTTGTTATTCGCCT TATCATTGACAGCGAGCTCA 4 TGGAGTCTGAAGAAGGACTC CATCTGGACTACGGCAACGA 6 AACTGTCATAAGACAGACAA 7 CCTCAACATGACATACACCG CAATACCGTTCGCGATTCTA GCGTCTACGTTGATTCGGCC TGAACAGAGGCACTTGCAGG H 1 CGACTAGAACCTACTACTGC 12 GCACCGCACGTGGAGAGATA 13 CTGAGAGACCGACTGATGCG GCGCGCTCGAAGTACAACAT 1293 14 TCGTCCTTCTACTTAATGAT AGATGCGTTGTTCC CAAGCTATACCATCCGAATT - GGAGCTCTGACCTGCAAGAA4:; ﬂ 16 CAATACGTATAGTCTTAGAT - AACATTAGCCTCAAGTAAGApp.00 17 CCATCCACAGTGACCTATGT - TGTGATTATGCCGAATGAGG1-7:-© 1297 18 TATCCGTTGGAGAAGGTTCA 19 CGCCTAGGTACCTGAGTACG CAGAGTGCTCGTGTTCGCGA 21 CGCTTGGACATCCTTAAGAA M2 GACCGCATGATTAGTCTTAC 23 CTTGGCCGTAGTCACTCAGT 4 GATAGCGATATTCAGTTCGC N) 5 CACTAAGACAACCA 57 6 CCATTCTGTTGCGTGTCCTC 27 ACATTCTGTACGCTTGCAGC 8 TGCTGAACGCCAATCGCTTA “—- 9 TCCTCTACAAGAATATTGCG CGACCAACGCAGCCTGATTC AGATGATGATCCAA 31 ATTGCGAGCTTGAGTAGCGC CTTGGATTCCAGGA 32 AAGGTGCGAGCATAGGAATC TGTTATAGCCACGATACTAT [Table 14—2] 72 TCGTCTCGACACAGTTGGTC 1320 73 TCCGTTCGCGTGCGAACTGA 1321 74 TCTGACTCTGGTGTACAGTC 1322 75 ACAGCGCAATTATATCCTGT 1323 76 GTACGTGAGACTAG 1324 77 TACATTGAAGCATCCGAACA 1325 78 CTCCTGAGAGATCAACGCCA 1326 79 TCACCTCGAATGAGTTCGTT 1327 TAGCGACTTAAGGTCCAAGC 1328 8 1 AGTACGTATTGCCGTGCAAG 1329 CTCCGATATCGCACGGATCG '87 AACTTATCGTCGGACGCATG 88 AATTCGTGCCGGTC ACAGCCTTCCTGTGTGGACT CCTCCGTGAGGATCGTACCA 91 GCTCTAAGTAACAGAACTAA 92 GACTTACCGCGCGTTCTGGT (.0 3 TCTGAGGATACACATGTGGA 94 TGTAATCACACTGGTGTCGG 95 CACTAGGCGGCAGACATACA CTAGAGCACAGTACCACGTT [Table 15—1] Table 15 List of random primers (22-base s) 7 Primer sequence SEQ ID NO l TTCAGAGGTCTACGCTTCCGGT 1345 2 GACTGCGTTATGCCAA 1346 3 TGCTGAGTTCTATACAGCAGTG 1347 4 ACCTATTATATGATAGCGTCAT 1348 ATCGTGAGCTACAGTGAATGCA 1349 CGTGATGTATCCGGCCTTGCAG 1350 7 TCTTCTGGTCCTAGAGTTGTGC 1351 TGATGTCGGCGGCGGATCAGAT 1352 TCGGCCTTAGCGTTCAGCATCC 1353 TTAAGTAGGTCAGCCACTGCAC 1354 .—A 1 CCAGGTGAGTTGATCTGACACC 1355 D—‘H 2 TATACTATTACTGTGTTCGATC 1356 3 CCGCAGTATGTCTAGTGTTGTC 1357 14 GTCTACCGCGTACGAAGCTCTC 1358 1 U1 ATGCGAGTCCGTGGTCGATCCT 1359 TGGTAGATTGGTGTGAGAACTA 1360 AGGTTCGTCGATCAACTGCTAA 1361 H CO ACGACAAGCATCCTGCGATATC 1362 TTGAATCACAGAGAGCGTGATT 1363 GTACTTAGTGCTTACGTCAGCT 1364 MN) 1 GATTATTAAGGCCAAGCTCATA 1365 2 GCATGCAGAGACGTACTCATCG 1366 MN 3 TAGCGGATGGTGTCCTGGCACT 1367 4 TACGGCTGCCAACTTAATAACT 1368 N5 TGACAACTTCTATAGT 1369 [\3 03 CAAGCAATAGTTGTCGGCCACC 1370 N7 TTCAGCAATCCGTACTGCTAGA 1371 [\D8 TGAGACGTTGCTGACATTCTCC 1372 29 GTTCCGATGAGTTAGATGTATA 1373 TTGACGCTTGGAGGAGTACAAG 1374 31 TTCATGTTACCTCCACATTGTG 1375 32 GAGCACGTGCCAGATTGCAACC 1376 [Table 15—2] 2 Primer sequence 33 GGTCGACAAGCACAAGCCTTCT 034 GGTAAGATGACCGACT 0003 5 CGAGGCATGCCAAGTCGCCAAT 6 GATAGGCGGATGAGAG 37 TTCGGTCTAGACCTCTCACAAT DD8 GTGACGCTCATATCTTGCCACC 039 GATGTAATTCTACGCGCGGACT 40 GATGGCGATGTTGCATTACATG 41 TATGCTCTGAATTAACGTAGAA 42 AGGCAATATGGTGATCCGTAGC AGGAGGATCCGTCAAGTCGACC 1401 AGAGTATGCCAGATCGTGAGGC 1402 CCACTCACTAGGATGGCTGCGT 1403 m TATCCAACCTGTTATAGCGATT 1404 TCTTGCAGTGAGTTGAGTCTGC 1405 CCACTGTTGTACATACACCTGG 1406 ATGCGCGTAGGCCACTAAGTCC 1407 ACAGCGGTCTACAACCGACTGC 1408 [Table 15—3] '2'. Primer sequence 65 TCGCGCTCCAGACAATTGCAGC CCGGTAGACCAGGAGTGGTCAT 67 ATCTCCTAACCTAGAGCCATCT CGAATCTAACAACTAC TAGTCTTATTGAATACGTCCTA 70 TCCTTAAGCCTTGGAACTGGCG .4 1 CCGTGATGGATTGACGTAGAGG a]2 GCCTGGATAACAGATGTCTTAG 3 CTCGACCTATAATCTTCTGCCA 4 TTCTCCTTCCTAATCA 75 ACACGCTATTGCCTTCCAGTTA 76 AAGCCTGTGCATGCAATGAGAA 7 TCGTTGGTTATAGCACAACTTC .48 GCGATGCCTTCCAACATACCAA 79 CCACCGTTAGCACGTGCTACGT GTTACCACAATGCCGCCATCAA 81 GGTGCATTAAGAACGAACTACC 82 TCCTTCCGGATAATGCCGATTC 83 AACCGCAACTTCTAGCGGAAGA Co 4 TCCTTAAGCAGTTGAACCTAGG 85 GTCAGATAAGATCAGA TTCGCCATAACTAGATGAATGC 87 AAGAAGTTAGACGCGGTGGCTG 88 GTATCTGATCGAAGAGCGGTGG TCAAGAGCTACGAAGTAAGTCC CGAGTACACAGCAGCATACCTA 91 CTCGATAAGTTACTCTGCTAGA 92 ATGGTGCTGGTTCTCCGTCTGT TCAAGCGGTCCAAGGCTGAGAC TGTCCTGCTCTGTTGCTACCGT AGTCATATCGCGTCACACGTTG m GGTGAATAAGGACATGAGAAGC [Table 16— 1] Table 16 List of random primers (24—base s) Primer sequence CCTGATCTTATCTAGTAGAGACTC TTCTGTGTAGGTGTGCCAATCACC GACTTCCAGATGCTTAAGACGACA GTCCTTCGACGGAGAACATCCGAG 1444- 1467 wo 2018/003727 [Table 16—2] 0 Primer sequence SEQ ID NO 33 TAGTAACCATAGCTCTGTACAACT 1473 0.1004 CGTGATCGCCAATACACATGTCGC 1474 TAATAACGGATCGATATGCACGCG 1475 036 ATCATCGCGCTAATACTATCTGAA 1476 37 CACGTGCGTGCAGGTCACTAGTAT 1477 W8 AGGTCCAATGCCGAGCGATCAGAA 1478 39 CAGCATAACAACGAGCCAGGTCAG 1479 40 ATGGCGTCCAATACTCCGACCTAT 1480 41 ATCGTGAATAATGAAGAC 1481 2 TCTCGACGTTCATGTAATTAAGGA 1482 g.»3 TCGCGGTTAACCTTACTTAGACGA 1483 4 ATCATATCTACGGCTCTGGCGCCG 1484 A}. 5 GCAGATGGAGACCAGAGGTACAGG 1485 6 AGACAGAAGATTACCACGTGCTAT 1486 E.km7 CCACGGACAACATGCCGCTTAACT 1487 8 CTTGAAGTCTCAAGCTATGAGAGA 1488 ACAGCAGTCGTGCTTAGGTCACTG 1489 AGGTGTTAATGAACGTAGGTGAGA 1490 AGCCACTATGTTCAAGCCTGAGCC 1491 52 GCAGGCGGTGTCGTGTGACAATGA 1492 AGCCATTGCTACAGAGGTTACTTA 1493 01 A}. ACAATCGAACCTACACTGAGTCCG 1494 CCGATCTCAATAGGTACCACGAAC 1495 6 GATACGTGGCGCTATGCTAATTAA 1496 7 AGAGAGATGGCACACATTGACGTC 1497 58 CTCAACTCATCCTTGTAGCCGATG 1498 9 GTGGAATAACGCGATACGACTCTT 1499 G:0 ATCTACCATGCGAATGCTCTCTAG 1500 l ATACGCACGCCTGACACAAGGACC 1501 62 TCTCAGTGTGTAGAGTCC 1502 63 AATATATCCAGATTCTCTGTGCAG 1503 64 CCTTCCGCCACATGTTCGACAAGG 1504 [Table 16—3] —u Primer sequence 65 ACTGTGCCATCATCCGAGGAGCCA TCTATGCCGCTATGGCGTCGTGTA G)7 CGTAACCTAAGGTAATATGTCTGC G:8 TACTGACCGTATCAAGATTACTAA 9 TCATCGGAGCGCCATACGGTACGT 0 GCAAGAGGAATGAACGAAGTGATT 1 GGCTGATTGACATCCTGACTTAGT 2 AAGGCGCTAGATTGGATTAACGTA 3 GCTAGCTAGAAGAATAGGATTCGT 74 CAGGTGACGGCCTCTATAACTCAT 75 CAGGTTACACATACCACTATCTTC 76 TTGCTACGTACCGTCTTAATCCGT 81 1521 0000 3 TCCGGACACACGATTAGTAACGGA 1523 4 TACGAAGTACTACAGATCGGTCAG 1524 00 5 AATTGTCAGACGAATACTGCTGGA 1525 TGAATCATGAGCCAGAGGTTATGC 1526 00 7 CACAAGACACGTCATTAACATCAA 1527 88 GAATGACTACATTACTCCGCCAGG 1528 AGCCAGAGATACTGGAACTTGACT 1529 TATCAGACACATCACAATGGATAC 1530 91 CTAGGACACCGCTAGTCGGTTGAA 1531 92 CTGCGTGTCCTGGTGTAT 1532 93 ATGCAATACTAAGGTGGACCTCCG 1533 94 ATGCAGACGCTTGCGATAAGTCAT 1534 95 TTGCTCGATACACGTAGACCAGTG 1535 TACTGGAGGACGATTGTCTATCAT 1536 {Table 17—1] Table 17 List of random primers (26-base primers) Z0 Primer sequence SEQ ID NO 1 ACTAAGGCACGCTGATTCGAGCATTA 1537 2 CGGATTCTGGCACGTACAAGTAGCAG 1538 3 TTATGGCTCCAGATCTAGTCACCAGC 1539 4 CATACACTCCAGGCATGTATGATAGG 1540 AGTTGTAAGCCAACGAGTGTAGCGTA 1541 GTATCAGCTCCTTCCTCTGATTCCGG 1542 7 AACATACAGAATGTCTATGGTCAGCT 1543 GACTCATATTCATGTTCAGTATAGAG 1544 AGAGTGAACGAACGTGACCGACGCTC 1545 AATTGGCGTCCTTGCCACAACATCTT 1546 ll TCGTAGACGCCTCGTACATCCGAGAT 1547 12 CCGGCTCGTGAGGCGATAATCATATA 1548 3 AGTCCTGATCACGACCACGACTCACG 1549 14 CAATCCTCCATGGAGAAGCT 1550 I—l 5 TCATCATTCCTCACGTTCACCGGTGA 1551 1 G] TCAACTCTGTGCTAACCGGTCGTACA 1552 17 TGTTCTTATGCATTAATGCCAGGCTT 1553 18 CGACCTCAACAGCATCACTC 1554 19 GGCGAGTTCGACCAGAATGCTGGACA 1555 TTCCGTATACAATGCGATTAAGATCT 1556 MN l GAGTAATCCGTAACCGGCCAACGTTG 1557 2 CGCTTCCATCATGGTACGGTACGTAT 1558 N3 CCGTCGTGGTGTGTTGACTGGTCAAC 1559 4 TATTCGCATCTCCGTATTAGTTGTAG 1560 TATTATTGTATTCTAGGCGGTGCAAC 1561 [\36 AGGCTGCCTACTTCCTCGTCATCTCG 1562 27 GTAACATACGGCTCATCGAATGCATC 1563 N8 CACGGATATTACCGTACGCC 1564 9 ATAGCACTTCCTCTAATGCTCTGCTG 1565 O TCACAGGCAATAGCCTAATATTATAT 1566 31 GGCGGATGTTCGTTAATATTATAAGG 1567 32 TGCAATAGCCGTTGTCTCTGCCAGCG 1568 [Table 17—2] 39 GAGACTGTTCAAGCTTGCTGTAGGAG 1575 TAACCGGAACTCGTTCAGCAACATTC 1576 41 TCAATTATGCATGTCGTCCGATCTCT 1577 42 TTGTCTAAGTCAACCTGTGGATAATC 1578 48 AAGGCTGCGATGAGAGGCGTACATCG 1584 49 GGTTCATGGTCTCAGTCGTGATCGCG 1585 50 TAGTGACTCTATGTCACCTCGGAGCC 1586 51 ATGTGATAGCAATGGCACCTCTAGTC 1587 0'1 2 AGTGTAATGCATCATCCGCT 1588 3 ATGTGGCGACGATCCAAGTTCAACGC 1589 4 ACCTTGTATGAGTCGGAGTGTCCGGC 1590 ACCTCAAGAGAGTAGACAGTTGAGTT 1591 6 GGTGTAATCCTGTGTGCGAAGCTGGT 1592 57 ATAGCGGAACTGTACGACGCTCCAGT 1593 58 AAGCACGAGTCGACCATTAGCCTGGA 1594 59 ATTCCGGTAACATCAGAAGGTACAAT 1595 GTGCAACGGCAGTCCAGTATCCTGGT 1596 61 TATACACGGTGACCGAAGAT 1597 62 GCACTTAATCAAGCTTGAGTGATGCT 1598 63 AGTATTACGTGAGTACGAAGATAGCA 1599 64 TTCTTAGGTTAAGTTCCTTCTGGACC 1600 [Table 17—3] SEQ ID NO GTCCTTGCTAGACACTGACCGTTGCT 1601 - ATGTGTGCTGCATCCTAAGC66 1602 67 CCATCAATAACAGACTTATGTTGTGA 1603 CGCGTGTGCTTACAAGTGCTAACAAG 1604 CGATATGTGTTCGCAATAAGAGAGCC 1605 70 CGCGGATGTGAGCGGCTCAATTAGCA 1606 71 GCTGCATGACTATCGGATGGAGGCAT 1607 ﬁll-4"}2 CTATGCCGTGTATGGTACGAGTGGCG 1608 3 CCGGCTGGAGTTCATTACGTAGGCTG 1609 4 TGTAGGCCTACTGAGCTAGTATTAGA 1610 CCGTCAAGTGACTATTCTTCTAATCT 1611 H.) 6 GGTCTTACGCCAGAGACTGCGCTTCT 1612 77 CGAAGTGTGATTATTAACTGTAATCT 1613 78 GCACGCGTGGCCGTAAGCATCGATTA 1614 79 ATCCTGCGTCGGAACGTACTATAGCT 1615 AGTATCATCATATCCATTCGCAGTAC 1616 00 l AGTCCTGACGTTCATATATAGACTCC 1617 82 CTTGCAGTAATCTGAATCTGAAGGTT 1618 83 ATAACTTGGTTCCAGTAACGCATAGT 1619 004 GATAAGGATATGGCTGTAGCGAAGTG 1620 85 GTGGAGCGTTACAGACATGCTGAACA 1621 G) CGCTTCCGGCAGGCGTCATATAAGTC 1622 7 TTCTAACCTCTATAAGCCGA 1623 88 ACGATCTATGATCCATATGGACTTCC 1624 TGAAGCTCAGATATCATGCCTCGAGC 1625 AGACTTCACCGCAATAACTCGTAGAT 1626 91 AGACTAAGACATACGCCATCACCGCT 1627 92 TGTAGCGTGATGTATCGTAATTCTGT 1628 3 TGTGCTATTGGCACCTCACGCTGACC 1629 94 TGTAGATAAGTATCCAGCGACTCTCT 1630 95 CCAATTGTGTGTAGGCGCAA 1631 CGATTATGAGTACTTGTAGACCAGCT 1632 [Table 18— 1] Table 18 List of random primers se primers) 16 1656 16 1651 26 1652 21 1653 22 1651 23 1655 26 1656 1653 26 1656 N7 1653 1666 [\D6 1661 36 1662 31 1663 32 1665 [Table 18—2] Primer sequence SEQ ID NO -33 CCAGTCCGGTGTACTCAGACCTAATAAC -34 CGAGACACTGCATGAGCGTAGTCTTATT 1666 TTGTATACTTCTCTACGGTCTG TTAGCTGGATGGAAGCCATATTCCGTAG -37 CAGCCTACACTTGAT'I‘ACTCAACAACTC -38 GTACGTAGTGTCACGCGCCTACGTTCGT -39 CTACAACTTCTCAATCATGCCTCTGTTG m CGAGGACAGAATTCGACATAAGGAGAGA GCCGAACGACACAGTGAGTTGATAGGTA GAACACTATATGCTGTCGCTGTCTGAGG GTTAAGTTCTTCGGCGGTCATGCTCATT TTGCTTACAGATCGCGTATCCATAGTAT E(II GAGGACCACCTCTGCGAAGTTCACTGTG AATCCTAGCATATCGAGAACGACACTGA TGAATACTATAGCCATAGTCGACTTCCG GACATCCACGAAGCTGGTAATCGGAACC TTAGCCGTCTTAGAAGTGTCTGACCGGC CTATTCTGCCGTAATTGATTCCTTCGTT ACGCCTCTGGTCGAAGGTAGATTAGCTC ATTGATCGTAAGTAGATGGTCC TTAAGTGAGGTGGACAACCATCAACTTC AAGGCCTTGCGGCTAAGTAGTATTCATC TTGTGATACTAATTCTTCTCAAGAGTCA GCATTAGGTGACGACCTTAGTCCATCAC GCGGATGGACGTATACAGTGAGTCGTGC I01 GAACATGCCAGCCTCAACTAGGCTAAGA TCCGTCATTAGAGTATGAGTGACTACTA m TAGTAACCAGTTCGGACTGGAC CGCTAACTATTGCGTATATTCGCGGCTT GCCATCTACGATCTTCGGCTTATCCTAG a03 CCTGAGAATGTTGACTAAGATCTTGTGA 1695 TCGGTTAGTCTAATCATCACGCAACGGA 1696 WO 03727 [Table 18—3] 6) 5 «34 123 --.]3 --J 5 OO 3 [Table 19—1] Table 19 List of random s (29-base primers) Primer sequence SEQ ID NO l CTCCTCGCCGATTGAAGTGCGTAGAACTA 1729 2 CAGCAGGCCTCAATAGGATAAGCCAACTA 1730 3 GACCATCAATCTCGAAGACTACGCTCTGT 1731 4 GGTTGCTCCGTCTGTTCAGCACACTGTTA 1732 AATGTCGACTGGCCATTATCGCCAAGTGT 1733 TTGCCATGCGAATGGATCTCCAG 1734 7 CCAGACCGGAGCCAATTGGCTGCCAATAT 1735 GCTCCATACGTTACCTAATGCAG 1736 GAATATGACGCGAACAGTCTATTCGGATC 1737 GACGAGAATGTATTAAGGATAAGCAAGGT 1738 11 AAGTCGTATGAATCGCTATCACATGAGTC 1739 1 GTCGTGGAGACTACAATTCTCCTCACGTT 1740 13 GTTGCCACCGTTACACGACTATCGACAGT 1741 AGGATAGGCTACGCCTTACTCTCCTAAGC 1742 H 01 TAATCATCCTGTTCGCCTCGAGGTTGTTA 1743 GACAAGCAGTAATAATTACTGAGTGGACG 1744 TACAGCGTTACGCAGGTATATCAAGGTAG 1745 ._‘ 00 CTAACATCACTTACTATTAGCGGTCTCGT 1746 CCGCGCTTCTTGACACGTTCTCCACTAGG 1747 CAAGTAACATGAGATGCTATCGGTACATT 1748 N 1 CGACCACTAGGCTGTGACCACGATACGCT 1749 22 CAGGTCATGTGACGCAGTCGGCAGTCAAC 1750 23 ACTCCATCGTTAGTTCTTCCGCCGTGCTG 1751 24 CTCACCACGTATGCGTCACTCGGTTACGT 1752 TGCCTATGCTATGGACCTTGCGCGACTCT 1753 6 AATGAAGGTCAACGCTCTGTAGTTACGCG 1754 [\3 7 CACCATTGATTCATGGCTTCCATCACTGC 1755 28 GACACGCAAGGTAATTCGAGATTGCAGCA 1756 [\D 9 CACCGAGAGGAAGGTTCGATCGCTTCTCG 1757 DJ O CAGTTATCGGATTGTGATATTCACTCCTG 1758 31 ATACTGTAACGCCTCAACCTATGCTGACT 1759 32 ATCTGTCTTATTCTGGCACACTCAGACTT 1760 2017/023343 [Table 19—2] 51 ATCAGATCTACTGATCGCGGTAGAGTATC 1779 52 TACACATAGGCGGCGCAGCCTTCTAATTA 1780 53 TTAACCGTAGTTCTTAGCTTACGCCGCTC 1781 54 ACTATAGAGGACATGGCACTCCTCTTCTA 1782 55 CAGTTCGTATTAAGATTGAATGTAGCGGT 1783 56 AGTTATCGGTATCCGCTTATCCGTACGTA 1784 57 AGCTTATTCATACACTGCACCACAGCAAG 1785 58 CCGTCGGCTAGTCTATCCTCTAATTAGAA 1786 59 GTCCGCTTCCATGCCTGCTGTACGAACAC 1787 TCTCTTCCTCCTTCATTGTTCGCTAGCTC 1788 61 TCTCTTGAGCGGTCCTCATACAGGTCTGC 1789 62 GACCAAGTGTAGGTGATATCACCGGTACT 1790 63 AAGATTGTGATAGGTTGGTAGTTACCACA 1791 64 TCGCCTCCGAAGAGTATAGCATCGGCAGA 1792 W0 2018/003727 [Table 19—3] O Primer sequence SEQ ID NO 65 GAGGTAGTTATGAGCATCGAGGTCCTGTT 1793 GGACGCAAGATCGCAGGTACTTGTAAGCT 1794 67 ACTCGTACACGTCATCGTGCAGGTCTCAG 1795 GTCAGGAGTGAGATGGCTCGACA 1796 AAGATGGTTCCGCGCATTGACTAGCAAGT 1797 70 TCCGCGATCTGCGGATCTTGAATGCTCAC 1798 71 TTCACGAGAGTCAACTGCTAGTATCCTAG 1799 ---J2 TTCCAACTGGATTCTTCCAACTCCTCGAA 1800 3 CACTACTACTCAAGTTATACGGTGTTGAC 1801 -.;| I-D CAACTGGATTCTCAGGATGCGTCTCTAGC 1802 75 AGAGTGGAGCGATTACGTAATAT 1803 76 GAGGTCATTCAACTGGACTCGCCACGGAC 1804 *4 7 TGTAACGCTGCAATCACATGAAT 1805 78 TATGCTGAGGTATTAGTTCTAACTATGCG 1806 79 CGTCTGAGTCGGATAAGGAAGGTTACCGC 1807 GTACTATCGTCGCAGGCACTATCTCTGCC 1808 81 GCTTCCTCCTTGCAACTTCATTGCTTCGA 1809 oo2 TGTCTACGAAGTAGAAGACACGAATAATG 1810 000:) 00 CCGTCATCTAAGGCAGAGTACATCCGCGA 1811 pb CCGGAGGCGTACTAACTGACCACAACACC 1812 85 AACTCGTCGCTGCCTGAATAGGTCAGAGT 1813 TTATAAGATTAATGTCGGTCAGTGTCGGA 1814 87 CGTCTCGATGGATCCACACGAACCTGTTG 1815 88 ATGCCATCATGGTCGTCCTATCTTAAGGC 1816 GCGCTTCAGCGATTCGTCATGCAAGGCAC 1817 CCAAGCGATACCGAGGTACGGTTAACGAG 1818 91 ATATGACAGACAGGTGGACCTAAGCAAGC 1819 92 CACTACATCGTCAGGCCTGGAAGCCTCAG 1820 (D 3 GCCGTGTAGACGAGGACATTATGTCGTAT 1821 (D4 CAACGTATATACACACCTTGTGAAGAGAA 1822 95 TCCAACGTAATTCCGCCGTCTGTCGAGAC 1823 AATTCGTGCTTCGATCACCGTAGACTCAG 1824 [Table 20— 1] Table 20 List of random primers (30-base primers) O SEQ ID No —1828 2 1828 8 1827 4 1828 8 1828 1888 1 1881 1882 1888 1881 11 1888 22 TCTCTACACAGCTACATACTATACTGTAAC 1846 23 TACGACGGACGCTGGTGGTGTAAGAGAAGG 1847 24 ATATATCTACGTATAGTTCAAGTT 1848 GGCTCCTGCATTCATTGAAGGTCGGCCTTG 1849 N6 CAGTTCGGTGATTCAAGAGAACAATGGTGG 1850 7 TATAACGAAGCCGGCTGGAACGGTAACTCA 1851 NM 8 CTGTATCAATTCAAGTGACAGTGGCACGTC 1852 9 AGCAATTGCGGTTCATAGGCGTAATTATAT 1853 CATATGGACCTGGAGATCACCGTTCAGTCC 1854 31 GAAGGCCGTTGGTCTATCTCTTACTGGAGC 1855 32 GTGCGTTCATCTAGCCTAAGACGCTGACCT 1856 WO 03727 [Table 20—2] [Table 20—3] __77 78 TGTATTATCTCGAAGCGGTGCGTTAGAGTC 1902 79 TTCTAGCTACTAATGGCGTCAATT 1903 CGCGCTACATTACTTCCTACACCATGCGTA 1904 81 TGAGGCAACTAGTGTTCGCAAGATGACGGA 1905 82 TTATTATTGTCTGTGGAACGCACGCCAGTC 1906 CO 3 GCTATAGTATTATCCATGAATTCCGTCGGC 1907 84 GTATCAATAGCTCAATTCGTCAGAGTTGTG 1908 85 TAGTCCATGCGTGGATATATTGAGAGCTGA 1909 GCACAGTACGACTTATAACAGGTCTAGATC 1910 87 ACTCAATGGTGGCACGCTCGGCGCAGCATA 1911 88 GTAGTACCACTCCGCCTTAGGCAGCTTAAG 1912 CGCTCAACTGATGCGTGCAACCAATGTTAT 1913 GCAGCTTGACTGCCTAGACAGCAGTTACAG 1914 91 GCAACTTCTTAGTACGAATTCATCGTCCAA 1915 ED2 ATGCTGCGGCAGTGGAGGTGGCTT 1916 3 TGCGGATCAATCCAGTTCTGTGTACTGTGA 1917 (04 TTATGATTATCACCGGCGTAACATTCCGAA 1918 95 GCTACCTAGATTCTTCAACTCATCGCTACC 1919 CAGTGTTAGAATGGCGGTGTGTAGCCGCTA 1920 [Table 21—1] Table 21 List of random s (3 5-base primers) O SEQ ID No _1921 2 1922 9 1923 9 1929 9 1925 1929 1 1922 1929 1929 ATATCGCGAGCACTAACGTCGTTGTCGTTCTAGGA 1930 GGTGGTCTGACCATAGCTGTTCTTCTCACAGAGAC 1940 21 GCAATACCAACGAGATGAGTATTCGTTGAAGCTCT 1941 22 CCAAGTCGACGCTGCATGAATGAGCGCTATTCACT 1942 23 CCATTAGATCGCTTCGAGACAATTAGGAGACATGA 1943 24 GATGACTGTACCTCCTATCATTGAGTGTGGACCAA 1944 ATATCTGGATGAATAGTGGTTAGGTAAGCAAGTAA 1945 26 ACCGACTATGTTAATTCGTGTCTGGATGGCAGAAT 1946 27 GTGGCAGTCTTGCTAGTATCTTAGACCATCACCAA 1947 28 CGCTATCTTAGTCGAGCACAATGTCTTCGTATAGG 1948 29 ATTAGTACGGCACGAACCGGCCATTCATGGCAGCT 1949 AGTACGACTATCAAGACTCCAGCGCTCTCCTTGGA 1950 ATGAGCCTCGGAGCGAACGTTATCGATCAGGCTGT 1951 TTGCGTGCAGTAGCACCGATACACAGCGCTTGTAT [Table 21—2] SEQ ID NO 33 1353 33 1353 1355 33 1353 33 1357 48 ACTCTACGGTGCACCTCAGCCTTCATGCAATAGGC 1968 49 CTTGTAGCACAATACATTACTCTCCACGTGATAGC 1969 50 TATCGATACCGTTATTCCTACTCTGTCGG 1970 51 GGATGATCGTCAACGATCAACTGACAGTTAGTCGA 1971 52 TGACAGTAGCAATGTCTCACGTCTGCACAACGGAA 1972 [Table 21—3] ZO Primer sequence 0}5 GGTTGCGATCAGCTTGATAGCAGGTCATATCCTCA 66 GCAGGTACTAACCTGAGATGCGTAGCTAACACAGG 6': 7 ATCTGCAAGGACGTAACGTCCTCGGAAGGTGAGGT 68 ATAATCTTACGAGCCTCCAGTGAATAATGCAAGCA G: 9 CAATCTCCGCACAGTCTTGTTCAGGTACAGACTTA 4-4O ATGTGCGCAATTCAGCGTAAGTGCCTATTCATAAT 1 TCGGACGCACACATCCTGTTGTCGAGAAGAGGAAG 2 TCGGAAGCATCACATGAGCATCAGGAGTTCATTGC 73 ATCTGGTTGTGGACTTCTATACAGTACCAGAGTGG 4 CGTCTGAATATAGTTAGCTAGTAGTGTAATCCAGG -..;| 5 TAATATCTGATCCGACCTATTATCTAGGACTACTC 76 TATGCGGCCGTCCGTACCTCGTCTGCTTCAGTTGG 77 TGGCTCAAGTTCCATATTGCCAAGACGACCTGGAG 78 GCAGTTCTGCTAGGCGGTCCGAGGCAATTGAAGAG 79 CATGGCACAGACGAAGTATGCACCACGCTCATTAA 1999 GGAGCGTACTACGACCATTCAACCGAATATGTTAC CO I ATCTCGCGACAGAGACAAGGTGCGAATGG oo 2 TGGACTGAGGTTCTCCGGTCTATACTCCTGTAGGA 3 TAGCAACGGCTTCTTGTGATCGCATTGCA 84 GGCGAAGAATCATGCGAGACGGAGTAGACGGACGT 85 GAGCATTGCGAGTTGCACACGTGATATCAGACTGT CTGTTGACCTATGCCAGAATCAATACCTCAGATTA 87 GTTAACAAGTAGATGCCAAGATACAACGAGAGACC 88 GATTATAGTTAGGAAGATAGTTAACTCGC TCCGGAGTCGAGCATATGTGACCAACTCTCAACGC GGAGCTGCGATGCCGTTACCGACGTCATCTTCAAG 91 GCTCTATCTTACACATTGGCGTACTGGACTCGCGA 92 TTCTACATATTCATCGCCTACCGAGTTGCGCGAAG 93 TGGACGTCTGACCTGTGTCTACATCGGTGGTGCTA 94 GGCAGGACAGCTCCGTG'I‘TCTACTCGAACCGCACT 95 TGACAACCTCATGTCTCCGACCGCAGGCATACAAT GCAGGCCTAACAAGTGGTCACGAGGAGTCCTTATT 2016 3.1.2 Standard PCR To the genomic DNA described in 2. above (15 ng, NiFS—derived genomic DNA), random primers (final concentration: 0.6 microM; 10—base primer A), a 0.2mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase STAR, TAKARA) 2017/023343 were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal cycling conditions sing 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. In this example, numerous c acid fragments obtained via PCR using random primers, including the standard PCR described above, are referred to as DNA libraries. 3.1.3 Purification of DNA library and electrophoresis The DNA library ed in 3.1.2 above was purified with the use of the MinElute PCR cation Kit (QIAGEN) and ted to electrophoresis with the use of the Agilent 2100 bioanalyzer (Agilent Technologies) to obtain a ﬂuorescence unit (FU). 3.1.4 Examination of ing ature To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers (final concentration: 0.6 microM; 10—base primer A), a 0.2mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, ) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 s, various ing temperatures for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. In this example, annealing temperature of 37 degrees C, 40 degrees, and 45 degrees C were examined. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.1.5 Examination of enzyme amount To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers (final concentration: 0.6 microM; 10—base primer A), a 0.2mM dNTP mixture, 1.0 mM MgC12, and 2.5 units or 125 units of DNA polymerase STAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. The DNA library ed in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.1.6 ation of MgClz concentration To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers (final concentration: 0.6 microM; 10—base primer A), a 0.2mM dNTP mixture, MgClz at a given concentration, and 1.25 units of DNA polymerase STAR, ) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under l cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, ed by storage at 4 degrees C. In this example, MgClz concentrations, which are 2 times (2.0 mM), 3 times (3.0 mM), and 4 times (4.0 mM) greater than a common level, respectively, were examined. The DNA library obtained in this experiment was subjected to cation and electrophoresis in the same manner as in 3.1.3. 3.1.7 Examination of base length of random primer To the c DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers (final concentration: 0.6 microM), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 mi— croliters. PCR was d out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 s, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. In this example, the random primers comprising 8 bases (Table 7), 9 bases (Table 8), 11 bases (Table 9), 12 bases (Table 10), 14 bases (Table 11), 16 bases (Table 12), 18 bases (Table 13), and 20 bases (Table 14) were examined. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.1.8 Examination of random primer concentration To the c DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers at a given concentration (10—base primer A), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was ed while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal g ions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by e at 4 degrees C.

In this example, random primer concentrations of 2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 microM were examined. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. In this experiment, the reproducibility of the repeated data was ted on the basis of the Spearman's rank correlation (rho > 0.9). 3.2 Verification of reproducibility via MiSeq 3.2.1 Preparation of DNA library To the c DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers (final tration: 60 microM, 10—base primer A), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under l cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 s C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.2.2 Preparation of sequence library From the DNA library obtained in 3.2.1, a sequence library for MiSeq analysis was prepared using the KAPA Library Preparation Kit ). 3.2.3 MiSeq analysis With the use of the MiSeq Reagent Kit V2 500 Cycle (Illumina), the sequence library for MiSeq analysis obtained in 3.2.2 was ed via 100 base paired—end sequencing. 3.2.4 Read data analysis Random primer sequence information was deleted from the read data obtained in 3.2.3, and the read patterns were fied. The number of reads was counted for each read pattern, the number of reads of the repeated analyses, and the reproducibility was evaluated using the correlational coefficient. 3.3 Analysis of rice y Nipponbare 3.3.1 Preparation of DNA library To the genomic DNA described in 2. above (15 ng, Nipponbare—derived genomic DNA), a random primer (final concentration: 60 microM, (10—base primer A), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was ed while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal cycling conditions sing 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. The DNA library obtained in this experiment was subjected to purification and ophoresis in the same manner as in 3.1.3. 3.3.2 Preparation of sequence library, MiSeq is, and read data analysis Preparation of a sequence library using the DNA library prepared from Nipponbare— derived genomic DNA, MiSeq analysis, and analysis of the read data were performed in accordance with the methods bed in 3.2.2, 3.2.3, and 3.2.4, respectively. 3.3.3 Evaluation of genomic homogeneity The read patterns obtained in 3.3.2 were mapped to the genomic information of Nipponbare (NC_008394 to NC_008405) using , and the genomic ons of the read patterns were identified. 3.3.4 Non—specific amplification On the basis of the positional ation of the read patterns identified in 3.3.3, the sequences of random primers were compared with the genome sequences to which such random primers would anneal, and the number of mismatches was determined. 3.4 Detection of polymorphism and identification of genotype 3.4.1 Preparation of DNA library To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA, Ni9—derived genomic DNA, hybrid progeny—derived genomic DNA, or Nipponbare— derived genomic DNA), random primers (final tration: 60 microM, 10—base primer A), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was d out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. The DNA y obtained in this ex— periment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.4.2 HiSeq analysis Analysis of the DNA ies prepared in 3.4.1 was consigned to TakaraBio under conditions in which the number of samples was 16 per lane via 100 base paired—end se— quencing, and the read data were obtained. 3.4.3 Read data is Random primer sequence information was deleted from the read data obtained in 3.4.2, and the read ns were identified. The number of reads was counted for each read pattern. 3.4.4 Detection of polymorphism and identification of genotype On the basis of the read patterns and the number of reads ed as a results of analysis conducted in 3.4.3, polymorphisms peculiar to NiF8 and Ni9 were detected, and the read patterns thereof were designated as markers. On the basis of the number of reads, the genotypes of the 22 hybrid progeny lines were identified. The accuracy for genotype fication was evaluated on the basis of the reproducibility attained by the repeated data concerning the 22 hybrid progeny lines. 3.5 Experiment for confirmation with PCR marker 3.5.1 Primer designing Primers were ed for a total of 6 s (i.e., 3 NiF8 markers and 3 Ni9 s) among the markers identified in 3.4.4 based on the marker sequence in— formation ed via paired—end sequencing (Table 22).

[Table 22] e<o<ohoo<¢o<$o B 88555259 B 05.588528 B 9 B Q SEE 385:0 38 amomoz 05823 39 aqmomuoz REESE ommu @862 ou<ohho<8<ogo 855$: Se Ammomuoz ouophcoouhuhgu 9635: ommv @862 83:25:00 85.5% ommv “$862 B n: n: n: B E 3E5 <oo<o<o<u<e<oou 2.50:3 89 @802 303235504 05385 89 Ammomuoz 8 85:55 @832 85.850535 2 SE 35,588 3.9 "OZ e<o<o<eu<<ouuuo 0355.5 89 $2552 8865325 08:88 39 @362 85:63 Sing @532 @862 @362 :oﬂmEHomE acoueﬁqogﬁuogg808558: 5uew<<oe<u<uHou<<w¢ewoo<<u80.5 55:68ESESEEE ,5weu<<ooo<oeoﬁo<<uouoo<ﬂoop$ 950583525088250? EESESSWUSDEBO .550530805055220 :58qu @mownoz @862 B 823$SEQUSEESSEBSD B 55985058238855525 B 0% 39 H95.35uouuogzeoogoeﬁﬁodmzuo $6oou<¢owoougottcouhﬁooﬁuo hewugiuﬁgoweo B 0mg ommv SB:8Sooééoéoéouéoéa $3228582.2085888 oEBEOEBBUSEEUE B ¢EBéUESEEEESEEE 553$:EQSESEUES B ommv H<8uE$885825288202 95 SEEM 8 3 9:30:me MO; 8:263 was 8x52 E<§u$853528555558 $5ESEEEHEEDEEBH:92 2::<u<3<<u<u<ﬁipg<o @832 23354858885355556“ 55353583085502385 88:35EESSDEESO 3852 5%$54835:8535022 EEE2E535:03283225 EEEESSSBEBEB @862 smomuoz oz 3862 soumﬁuomzm B B B Unuﬁuhmm 39 9% 5.50we<9¢u§u$u¢<weuueu§uwugh 8.005uEOEwoyoeuoﬁohuoopoesg. gopoogooooﬁohous Qommv 80222850SBEBEESB E85$EOSBEOEEEBEE 8258288880588 Q ommv ommv @85535252525642 $658505585th4805 uEBEEEBuEﬁﬁEBE e 0% E oocoswom sends—Swag 3&qu 05:3.- NmZNmowz NS H332 a38% ammmmaz N338 oz smwﬁsz oomoswom mm nab 099 oEﬂ. whiz _@z his: WO 03727 3.5.2 PCR and electrophoresis With the use of the TaKaRa lex PCR Assay Kit Ver.2 (TAKARA) and the genomic DNA described in 2. above (15 ng, erived genomic DNA, Ni9—derived genomic DNA, or hybrid progeny—derived c DNA) as a template, 1.25 mi— croliters of Multiplex PCR enzyme mix, 12.5 microliters of 2x Multiplex PCR , and the 0.4 microM primer designed in 3.5.1 were added, and a reaction solution was prepared while adjusting the final reaction level to 25 microliters. PCR was carried out under thermal cycling conditions comprising 94 degrees C for 1 minute, 30 cycles of 94 degrees C for 30 seconds, 60 s C for 30 s, and 72 degrees C for 30 seconds, and retention at 72 degrees C for 10 s, followed by storage at 4 degrees C. The amplified DNA fragment was subjected to electrophoresis with the use of TapeStation (Agilent Technologies). 3.5.3 Comparison of genotype data On the basis of the results of electrophoresis obtained in 3.5.2, the pe of the marker was identified on the basis of the presence or absence of a band, and the results were compared with the number of reads of the marker. 3.6 ation between random primer density and length 3.6.1 Inﬂuence of random primer length at high concentration To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers of a given length (final concentration: 10 microM), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. In this experiment, the random primer lengths of 9 bases (Table 8), 10 bases (Table 1, 10—base primer A), 11 bases (Table 9), 12 bases (Table 10), 14 bases (Table 11), 16 bases (Table 12), 18 bases (Table 13), and 20 bases (Table 14) were examined. In the on system using a random primer of 9 bases, PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 37 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. In the reaction system using a random primer of 10 or more bases, PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 s C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by e at 4 degrees C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. 3.6.2 Correlation between random primer density and length To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), random primers of a given length were added to a given concentration therein, a 0.2 mM dNTP e, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added thereto, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. In this experiment, random primers comprising 8 to 35 bases shown in Tables 1 to 21 were examined, and the random primer concentration from 0.6 to 300 microM was examined.

In the on system using random primers each comprising 8 bases and 9 bases, PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 37 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. In the reaction system using a random primer of 10 or more bases, PCR was carried out under thermal g conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by e at 4 degrees C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3. 1.3. Also, the reproducibility of the repeated data was evaluated on the basis of the Spearman's rank correlation (rho > 0.9). 3.7 Number of random s To the genomic DNA bed in 2. above (15 ng, erived genomic DNA), 1, 2, 3, 12, 24, or 48 types of random primers selected from the 96 types of random primers comprising 10 bases (lO—base primer A) shown in Table l were added to the final concentration of 60 microM therein, a 0.2 mM dNTP mixture, 10 mM MgC12, and 1.25 units of DNA polymerase STAR, TAKARA) were added thereto, and a reaction solution was prepared while adjusting the final on level to 50 mi— ers. In this experiment, as the l, 2, 3, 12, 24, or 48 types of random primers, random primers were selected successively from No. 1 shown in Table l, and the selected primers were then examined. PCR was carried out under thermal cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 s C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C. The DNA library obtained in this experiment was ted to purification and electrophoresis in the same manner as in 3.13. Also, the repro— ducibility of the repeated data was evaluated on the basis of the Spearman’s rank cor— relation (rho > 0.9). 3.8 Random primer sequence To the genomic DNA described in 2. above (15 ng, NiF8—derived genomic DNA), a set of primers selected from the 5 sets of random primers shown in Tables 2 to 6 was added to the final concentration of 60 microM therein, a 0.2 mM dNTP mixture, 10 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, ) were added thereto, and a reaction solution was prepared while adjusting the final reaction level to 50 microliters. PCR was carried out under thermal g conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 degrees C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 degrees C.

The DNA library obtained in this experiment was subjected to purification and elec— trophoresis in the same manner as in 3.1.3. Also, the reproducibility of the repeated data was evaluated on the basis of the Spearman’s rank correlation (rho > 0.9). 3.9 DNA library using human—derived genomic DNA To the genomic DNA described in 2. above (15 ng, human—derived genomic DNA), a random primer (final concentration: 60 microM; 10—base primer A), a 0.2 mM dNTP mixture, 1.0 mM MgC12, and 1.25 units of DNA polymerase (PrimeSTAR, TAKARA) were added, and a reaction solution was prepared while adjusting the final reaction level to 50 iters. PCR was carried out under l cycling conditions comprising 98 degrees C for 2 minutes and 30 cycles of 98 s C for 10 seconds, 50 degrees C for 15 seconds, and 72 degrees C for 20 seconds, followed by storage at 4 s C. The DNA library obtained in this experiment was subjected to purification and electrophoresis in the same manner as in 3.1.3. Also, the ucibility of the repeated data was evaluated on the basis of the Spearman’ s rank ation (rho > 0.9). 4. Results and examination 4.1 Correlation between PCR conditions and DNA library size When PCR was conducted with the use of random primers in accordance with con— ventional PCR conditions (3.1.2 described above), the ied DNA library size was as large as 2 kbp or more, but amplification of the DNA library of a target size (i.e., 100—bp to 500—bp) was not observed (Fig. 2). A DNA library of 100 bp to 500 bp could not be obtained because it was highly unlikely that a random primer would function as a primer in a region of 500 bp or smaller. In order to prepare a DNA library of the target size (i.e., 100 bp to 500 bp), it was considered necessary to induce non—specific amplification with high reproducibility.

The correlation between the annealing temperature (3.1.4 above), the enzyme amount (3.1.5 above), the MgClz concentration (3.1.6 above), the primer length (3.1.7 above), and the primer concentration (3.18 above), which are considered to affect PCR specificity, and the DNA library size were ed.

Fig. 3 shows the s of the experiment described in 3.1.4 attained at an annealing ature of 45 s C, Fig. 4 shows the s attained at an annealing tem— perature of 40 s C, and Fig. 5 shows the results attained at an annealing tem— perature of 37 degrees C. By reducing the annealing temperature from 45 degrees C, 40 degrees C, to 37 degrees C, as shown in Figs. 3 to 5, the amounts of high— molecular—weight DNA y ied increased, although amplification of low— molecular—weight DNA library was not observed.

WO 03727 Fig. 6 shows the results of the experiment described in 3.1.5 attained when the enzyme amount is increased by 2 times, and Fig. 7 shows the results attained when the enzyme amount is increased by 10 times the original amount. By increasing the enzyme amount by 2 times or 10 times a common amount, as shown in Figs. 6 and 7, the amounts of high—molecular—weight DNA library amplified sed, although am— plification of lecular—weight DNA library was not observed.

Fig. 8 shows the results of the experiment described in 3.1.6 attained when the MgClz concentration is increased by 2 times a common amount, Fig. 9 shows the results attained when the MgClz concentration is increased by 3 times, and Fig. 10 shows the results attained when the MgClz concentration is increased by 4 times. By sing the MgClz concentration by 2 times, 3 times, and 4 times the common amount, as shown in Figs. 8 to 10, the amounts of high—molecular—weight DNA library amplified varied, although amplification of a low—molecular—weight DNA library was not observed.

Figs. 11 to 18 show the results of the experiment described in 3.1.7 ed at the random primer lengths of 8 bases, 9 bases, 11 bases, 12 bases, 14 bases, 16 bases, 18 bases, and 20 bases, respectively. Regardless of the length of a random primer, as shown in Figs. 11 to 18, no significant change was ed in comparison with the results shown in Fig. 2 (a random primer comprising 10 bases).

The results of experiment described in 3.1.8 are summarized in Table 23.

[Table 23] Concentration . Correlational Repeat Flg' NO' (H M) coefficient (p) 2 — Fig. 19 — 4 — Fig. 20 — First Fig. 21 6 0689 Second Fig. 22 First Fig. 23 8 0361 Second Fig. 24 First Fig. 25 0'9 ’9.- Second Fig. 26 First Fig. 27 0'950 Second Fig. 28 First Fig. 29 Second . O 975.

Flg. 30 First Fig. 31 60 0359 Second Fig. 32 First Fig. 33 100 0383 Second Fig. 34 First Fig. 35 200 .° 0 991 Second .

FIg. 36 First Fig. 37 300 0395 Second Fig. 38 First Fig. 39 400 0388 'Second Fig. 40 First Fig. 41 500 0371 Second Fig. 42 600 . — Fig. 43 — 700 — Fig. 44 — 800 — Fig. 45 — 900 — Fig. 46 — 1000 - Fig. 47 — With the use of random s comprising 10 bases, as shown in Figs. 19 to 47, am— plification was observed in a l—kbp DNA fragment at the random primer concentration of 6 microM. As the concentration increased, the molecular weight of a DNA fragment decreased. Reproducibility at the random primer concentration of 6 to 500 microM was examined. As a , a relatively low rho value of 0.889 was attained at the con— centration of 6 microM, which is 10 times higher than the usual level. At the con— tion of 8 microM, which is equivalent to 13.3 times higher than the usual level, and at 500 microM, which is 833.3 times higher than the usual level, a high rho value of 0.9 or more was attained. The results demonstrate that a DNA fragment of 1 kbp or smaller can be amplified while achieving high reproducibility by elevating the random primer concentration to a level significantly higher than the concentration employed under general PCR conditions. When the random primer tration is excessively higher than 500 iter, amplification of a DNA fragment of a desired size cannot be observed. In order to amplify a low—molecular—weight DNA fragment with excellent reproducibility, accordingly, it was found that the random primer concentration should fall within an optimal range, which is higher than the concentration ed in a general PCR procedure and equivalent to or lower than a given level. 4.2 Confirmation of reproducibility via MiSeq In order to m the reproducibility for DNA library production, as described in 3.2 above, the DNA library amplified with the use of the genomic DNA extracted from NiF8 as a template and random primers was analyzed with the use of a next—generation sequencer (MiSeq), and the results are shown in Fig. 48. As a result of 3.2.4 above, 47,484 read patterns were obtained. As a result of comparison of the number of reads obtained through repeated measurements, a high correlation (i.e., a correlational co— efficient "r" of 0.991) was obtained, as with the results of electrophoresis. Accordingly, it was considered that a DNA library could be produced with satisfactory repro— ducibility with the use of random primers. 4.3 Analysis of rice y Nipponbare As described in 3.3 above, a DNA y was prepared with the use of genomic DNA extracted from the rice variety Nipponbare, the genomic ation of which has been sed, as a template, and random primers and subjected to electrophoresis, and the results are shown in Figs. 49 and 50. On the basis of the results shown in Figs. 49 and 50, the rho value was found to be as high as 0.979. Also, Fig. 51 shows the results of analysis of the read data with the use of MiSeq. On the basis of the s shown in Fig. 51, the ational coefficient "r" was found to be as high as 0.992. These results demonstrate that a DNA library of rice could be produced with very high repro— ducibility with the use of random s.

As described in 3.3.3, the obtained read pattern was mapped to the genomic in— formation of Nipponbare. As a result, DNA fragments were found to be evenly amplified throughout the genome at intervals of 6.2 kbp (Fig. 52). As a result of comparison of the sequence and genome information of random primers, 3.6 mismatches were found on average, and one or more mismatches were ed in 99.0% of primer pairs (Fig. 53). The results demonstrate that a DNA library ing the use of random primers is produced with satisfactory reproducibility via non— specific amplification evenly throughout the genome. 4.4 Detection of polymorphism and genotype identification of ane As described in 3.4, DNA libraries of the sugarcane varieties NiF8 and Ni9 and 22 hybrid progeny lines were produced with the use of random primers, the resulting DNA libraries were analyzed with the next—generation sequencer (HiSeq), the poly— sms of the parent varieties were detected, and the genotypes of the hybrid progenies were identified on the basis of the read data. Table 24 shows the results.

[Table 24] q88.8 88.8 c88.8 N85 28.8 88,8 cosmumwuoﬂ :88 88.8 88.8 mmbouom «8.8 Mom $8.: 88,8 38:03 was 88.8 $88 88.8 80x35 1 9.8 088 08.: 08.8 333mg mamuimﬁ mo «48.8 mo 88.: 88.8 38:52 H8332 2an EEa As shown in Table 24, 8,683 NiFS s and 11,655 Ni9 mafkel‘S‘ that is, a total of WO 03727 ,338 markers, were produced. In addition, reproducibility for genotype identification of hybrid progeny lines was as high as 99.97%. This indicates that the accuracy for genotype identification is very high. In particular, sugarcane is polyploid (8x+n), the number of chromosomes is as large as 100 to 130, and the genome size is as large as Gbp, which is at least 3 times greater than that of humans. Accordingly, it is very difficult to fy the genotype throughout the genomic DNA. As described above, numerous markers can be produced with the use of random primers, and the sugarcane pe can thus be identified with high accuracy. 4.5 Experiment for confirmation with PCR marker As described in 3.5 above, the sugarcane ies NiF8 and Ni9 and 22 hybrid progeny lines were subjected to PCR with the use of the primers shown in Table 22, genotypes were identified via electrophoresis, and the results were compared with the number of reads. Figs. 54 and 55 show the number of reads and the electrophoretic pattern of the NiF8 marker N8052l 152, respectively. Figs. 56 and 57 show the number of reads and the electrophoretic pattern of the NiF8 marker N80997l92, respectively.

Figs. 58 and 59 show the number of reads and the electrophoretic pattern of the NiF8 marker N80533l42, respectively. Figs. 60 and 61 show the number of reads and the electrophoretic pattern of the Ni9 marker N9l55239l, respectively. Figs. 62 and 63 show the number of reads and the electrophoretic pattern of the Ni9 marker N9l653962, respectively. Figs. 64 and 65 show the number of reads and the elec— trophoretic pattern of the Ni9 marker N9l 124801, respectively.

As shown in Figs. 54 to 65, the s for all the PCR markers designed in 3.5 above were consistent with the results of analysis with the use of a next—generation sequencer.

It was thus considered that genotype identification with the use of a next—generation sequencer would be able as a marker technique. 4.6 Correlation between random primer density and length As described in 3.6. l, the results of DNA library production with the use of random s comprising 9 bases (Table 8), 10 bases (Table l, lO—base primer A), ll bases (Table 9), 12 bases (Table 10), 14 bases (Table ll), 16 bases (Table l2), 18 bases (Table 13), and 20 bases (Table 14) are shown in Figs. 66 to 81. The s are summarized in Table 25.

[Table 25] Random primer Correlational Repeat Fig. No. length coefﬁcient (9) 9 85:23:. a: 2: w £22.22 F" ' t _, 11 sea; iii: ii 0'9” 12 85:25:. 3:92 F" 2:4:' ' t ." 14 3.22:.

F' £12.43- . _.— 16 3922:; “-989 18 85228;. £134: First Fig. 80 0.999 Second Fig. 81 When random primers were used at a high concentration of 10.0 , which is 13.3 times greater than the usual level, as shown in Figs. 66 to 81, it was found that a low—molecular—weight DNA nt could be amplified with the use of random primers comprising 9 to 20 bases while achieving very high reproducibility. As the base length of a random primer increased (12 bases or more, in particular), the molecular weight of the amplified fragment was likely to be decreased. When random primers comprising 9 bases were used, the amount of the DNA nt amplified was increased by setting the annealing temperature at 37 degrees C.

In order to elucidate the correlation between the density and the length of random primers, as described in 3.6.2 above, PCR was carried out with the use of random primers comprising 8 to 35 bases at the tration of 0.6 to 300 microM, so as to produce a DNA library. The results are shown in Table 26.

WO 03727 [Table 26] Correlation between concentration and length of random primer relative to DNA library Concentration Primer length relative to standard 01 DNA library covering 100 to 500 bases is ampliﬁed with good reproducibility (p > 0.9) X : DNA library not covering 100 to 500 bases or ucibility is poor (p S 0.9) '2 Unperfonned As shown in Table 26, it was found that a low—molecular—weight (100 to 500 bases) DNA nt could be amplified with high reproducibility with the use of random primers comprising 9 to 30 bases at 4.0 to 200 microM. In particular, it was confirmed that low—molecular—weight (100 to 500 bases) DNA fragments could be amplified assuredly with high reproducibility with the use of random primers comprising 9 to 30 bases at 4.0 to 100 microM.

The results shown in Table 26 are examined in greater detail. As a result, the cor— relation between the length and the tration of random primers is found to be preferably within a range surrounded by a frame as shown in Fig. 82. More specifically, the random primer concentration is preferably 40 to 60 microM when the random primers comprise 9 to 10 bases. It is able that a random primer con— centration satisfy the condition represented by an inequation: y > 3E + 08x69”, provided that the base length of the random primer is represented by y and the random primer concentration is represented by x, and 100 microM or lower, when the random primer comprises 10 to 14 bases. The random primer concentration is preferably 4 to 100 mM when the random primer comprises 14 to 18 bases. When a random primer comprises 18 to 28 bases, the random primer tration is preferably 4 microM or higher, and it satisfies the ion represented by an inequation: y < 8E +08X'5‘533.

When a random primer comprises 28 to 29 bases, the random primer tration is preferably 4 to 10 microM. The inequations y > 3E + 08x5974 and y < 8E +08x5-533 are determined on the basis of the Microsoft Excel power approximation.

By prescribing the number of bases and the concentration of random primers within given ranges as described above, it was found that low—molecular—weight (100 to 500 bases) DNA fragments could be amplified with high reproducibility. For example, the accuracy of the data obtained via analysis of high—molecular—weight DNA fragments with the use of a next—generation cer is known to deteriorate to a significant extent. As bed in this example, the number of bases and the concentration of random primers may be prescribed within given ranges, so that a DNA library with a molecular size le for analysis with a next—generation sequencer can be produced with satisfactory reproducibility, and such DNA library can be suitable for marker analysis with the use of a next—generation cer. 4.7 Number of random primers As described in 3.7 above, 1, 2, 3, 12, 24, or 48 types of random primers (concentration: 60 microM) were used to produce a DNA library, and the results are shown in Figs. 83 to 94. The results are summarized in Table 27.

[Table 27] Number of random . Correlational . Repeat Flg N0. . coefﬁment (p). prlmers First Fig. 83 1 0 984 Second Fig. 84 ' First Fig. 85 2 (1968 Second Fig. 86 First Fig. 87 3 0914,_ Second Fig. 88 First Fig. 89 12 0993 Second Fig. 90 First Fig. 91 24 0986 Second Fig. 92 Flrst Flg. 93 48 0' 978 Second Fig. 94 As shown in Figs. 83 to 94, it was found that low—molecular—weight DNA fragments could be amplified with the use of any of l, 2, 3, 12, 24, or 48 types of random primers while achieving very high reproducibility. As the number of types of random primers increases, in particular, a peak in the electrophoretic pattern lowers, and a ion is likely to ear. 4.8 Random primer sequence As described in 3.8 above, DNA ies were produced with the use of sets of random primers shown in Tables 2 to 6 (i.e., e primer B, lO—base primer C, —base primer D, lO—base primer E, and lO—base primer F), and the results are shown in Figs. 95 to 104. The results are summarized in Table 28.

[Table 28] Set of random primers Repeat Fig. No. CorrelatiOnal coeffiment (p) First Fig. 95 lO-base - prlmers B Second Flg. 96_ 0 916_ Fig. 97 lO-base - First ptimers C Second Flg. 98_ 0'965 - First Fig. 99 lO-base s D 0'986 Second Fig. 100 F1rst Fig. 101 lO-base s E 0.983 Second Fig. 102 lO-base . First Fig. 103 prlmersF . 0388 Second Fig. 104 As shown in Figs. 95 to 104, it was found that low—molecular—weight DNA fragments could be amplified with the use of any sets of 10—base primer B, 10—base primer C, —base primer D, 10—base primer E, or 10—base primer F while achieving very high re— producibility. 4.9 Production of human DNA library As described in 3.9 above, a DNA y was produced with the use of human— derived genomic DNA and random primers at a final concentration of 60 microM (10—base primer A), and the s are shown in Figs. 105 and 106. Fig. 105 shows the results of the first repeated experiment, and Fig. 106 shows the results of the second repeated experiment. As shown in Figs. 105 and 106, it was found that low— molecular—weight DNA fragments could be amplified while achieving very high repro— ducibility even if human—derived genomic DNA was used.

Example 2 In Example 2, a DNA probe was designed in accordance with the step schematically shown in Fig. 107, and a DNA microarray comprising the designed DNA probe was produced. In this example, r or not a DNA marker could be detected with the use of such DNA microarray was examined.

In this example, a DNA library was produced in the same manner as described in 3.2.1 of Example 1, except that the random primers comprising 10 bases shown in Table 1 and 30 ng of genomic DNAs of the sugarcane varieties NiF8 and Ni9 were used. In this example, also, a sequence library was produced in the same manner as bed in 3.2.2 of Example 1 and the ce library was ted to MiSeq analysis in the same manner as described in 3.2.3.

In this example, 306,176 types of DNA probes comprising 50 to 60 bases were designed on the basis of the sequence information of the DNA libraries of NiFS and Ni9 obtained as a result of MiSeq analysis, so as to adjust a TM at around 80 degrees C. The sequences of the designed DNA probes were compared with the sequence in— formation of NiFS and Ni9, and 9,587 types of probes peculiar to NiFS, which are not found in the Ni9 DNA library, and 9,422 types of probes ar to Ni9, which are not found in the NiFS DNA library, were selected. On the bases of a total of 19,002 types of the selected DNA probes, production of G3 CGH 8x60K Microarrays was consigned to Agilent Technologies, Inc.

With the use of the DNA microarrays thus produced, DNA libraries ed from NiFS, Ni9, and 22 hybrid progeny lines were subjected to detection.

DNA libraries of NiFS, Ni9, and 22 hybrid progeny lines were produced in the same manner as described in 3.2.1 of Example 1. Two DNA libraries were produced for Ni9 and for 2 hybrid y lines (i.e., Fl_01 and Fl_02), so as to obtain the repeated data.

The DNA libraries were ﬂuorescently labeled with the use of Cy3—Random Nonamers of the NimbleGen One—Color DNA Labeling Kit in accordance with the Gen Arrays User’s Guide.

With the use of the DNA microarrays and the fluorescently—labeled DNA libraries, subsequently, hybridization was carried out in accordance with the array—comparative genomic hybridization (array—CGH) method using the Agilent in—situ oligo—DNA mi— croarray kit. Subsequently, signals on the DNA microarrays when a relevant DNA library was used were detected with the use of the SureScan scanner.

On the basis of the signals ed for NiFS, Ni9, and 22 hybrid y lines, 7,140 types of DNA probes exhibiting clear signal intensities were identified. DNA fragments corresponding to such DNA probes can be used as NiFS markers and Ni9 markers. In this example, pe data were ed on the basis of signals obtained from DNA probes corresponding to the NiFS markers and the Ni9 markers, genotype data obtained through repeated measurements of two hybrid progeny lines (Fl_01 and Fl_02) were ed, and the accuracy for genotype identification was ted on the basis of the data reproducibility.

In this example, genotype data were obtained with the use of PCR markers in order to compare such data with the results of the DNA microarray experiment described above. Specifically, the primers described in 3.5 of Example 1 (Table 22) were used for the 3 NiFS markers and the 3 Ni9 markers described in 3.5 of Example 1 (i.e., a total of 6 markers). PCR and electrophoresis were performed in the manner as described in 3.5.2 of Example 1, and the s were compared with the signals obtained from the DNA microarray.

In this example, the DNA probes shown below were designed for the 6 markers shown in Table 22 (Table 29).

WO 03727 [Table 29] Marker DNA probe sequence name CACACACCATGAAGCTTGAACTAATTAACATTCTCAAACTAATTAACAAGCATGCAAGCA N80521 152 (SEQ ID NO:2041) CAAGTCCTCAATGTCATAGGCGAGATCGCAGTAGTTCTGTAACCATTCCCTGCTAAACTG N80997 l 92 (SEQ ID NO:2042) GTTTATCAAGATGGGTCATCGAGCTCTTGGTGTCTTCAACCTTCTTGACATCAACTTCTC N80533 142 (SEQ ID NO:2043) CTGAAGCTCTAGGTATGCCTCTTCATCTCCCTGCACCTCTGGTGCTAGCA N91552391 (SEQ ID 4) CTGTCTGCCATTGCCATGTGAGACAAGGAAATCTACTTCACCCCCATCTATCGA N9 1 653962 (SEQ ID NO:2045) TAAGATTAACTATGAACAAATTCACGGGTCCGATTCCTTTGGGATTTGCAGCTTGCAAGA N91 124801 (SEQ ID NO:2046) s and Examination DNA microarray analysis The sugarcane varieties NiFS and Ni9 and 22 hybrid progeny lines were analyzed with the use of the DNA microarray produced in the manner described above. As a result, 3,570 markers exhibiting apparently different signals between parent varieties were identified as shown in Table 30 (Fig. 108).

[Table 30] HII hﬁﬂﬂoswoagom $0o 02 $3? $8.8 E 0% 2; 0.2 . . m m or ﬁaﬂoswoammm Hx50 2: $8.03 $8.02 E 8m a H £3 Sam mﬁmﬁoswoamom £8.02 $3.8 33.8 E 33 :3 3% 282 mamuﬁada 33 mg; 83 I$34 25awormE ning Ni9, signals obtained through repeated procedures were compared, and a high correlation was found therebetween as a consequence (Fig. 109: r = 0.9989). On the basis of the results, the use of random primers at a high concentration was predicted to enable the production of a DNA y with excellent reproducibility and the use of a DNA probe was ted to enable the detection of a DNA fragment contained in a DNA library (i.e., a marker).

As a result of DNA microarray analysis using the 22 hybrid progeny lines, a total of 78,540 genotype data were obtained, and no missing values were observed for any markers. In order to evaluate the accuracy for genotype identification, the data obtained through repeated analyses of Fl_01 and those of F1_02 were compared. As a result, all the data concerning the NiF8 markers were consistent. Concerning the Ni9 , a result concerning Fl_01 was different, although all the results ning Fl_02 were consistent. With respect to all the markers, 7,139 data out of 7,140 pe data were consistent; that is, a very high degree of reproducibility was observed (i.e., the degree of consistency: 99.99%).

Experiment for ation with the use of PCR marker Concerning a total of 6 markers (i.e., 3 NiF8 markers and 3 Ni9 markers), s designed on the basis of the paired—end marker sequence information were used to subject NiF8, Ni9, and 22 hybrid progeny lines to PCR, the genotypes thereof were identified via ophoresis, and the results were ed with the signals obtained from the DNA microarray. Fig. 110 shows the results of measurement of signal levels obtained from the DNA probes corresponding to the marker (N80521152), Fig. 111 shows the results of measurement of signal levels ed from the DNA probes reacting with the marker (N80997192), Fig. 112 shows the results of measurement of signal levels obtained from the DNA probes reacting with the marker (N80533142), Fig. 113 shows the results of measurement of signal levels obtained from the DNA probes reacting with the marker (N91552391), Fig. 114 shows the results of mea— surement of signal levels obtained from the DNA probes ng with the marker (N91653962), and Fig. 115 shows the results of measurement of signal levels obtained from the DNA probes reacting with the marker (N91124801). Fig. 55 shows the elec— retic pattern for the marker (N80521152), Fig. 57 shows the electrophoretic pattern for the marker (N80997192), Fig. 59 shows the electrophoretic pattern for the marker (N80533 142), Fig. 61 shows the electrophoretic pattern for the marker (N91552391), Fig. 63 shows the electrophoretic pattern for the marker (N91653962), and Fig. 65 shows the electrophoretic pattern for the marker (N91124801). As a result of comparison of the results of electrophoretic patterns and the results of measurement of signal values obtained from DNA probes, the results for all s are found to be consistent among all the markers. The results trate that a DNA probe may be designed on the basis of the nucleotide sequence of the DNA fragment contained in the DNA library resulting from the use of a random primer at a high tration, so that the DNA fragment can be detected with high accuracy.

Trimmed: acial éEQl/JEHZDJZAOESAQQQ PCT/JP 2017/023 343 - 16.07.2018

Claims

[Claim 1] (Amended) ~ A method for producing a DNA probe comprising steps of: conducting a nucleic acid ampliﬁcation reaction in a reaction solution containing genomic DNA, Pfu DNA rase and a random primer having 9~30 bases at a high concentration using genomic DNA as a template to obtain DNA fragments, wherein when the random primer comprises 9 to 10 bases, the concentration of the random primer is 40 to 60 microM; when the random primer comprises 10 to 14 bases, the concentration of the random primer y the conditions deﬁned by an inequation: y > 3E + 08x'6’974 and be 100 microM or less, provided that the base length of the random primer is represented by "y" and the concentration of the random primer is represented by "x"; when the random primer comprises 14 to 18 bases, the concentration of the random primer is 4 to 100 microM; when the random primer comprises 18 to 28 bases, the concentration of such random primer be 4 microM or more and satisfy the conditions deﬁned by an inequation: y < 8E + 0816“”; when the random primer comprises 28 to 29 bases, the concentration of the random primer is 6 to 10 microM; when the random primer comprises 30 bases, the tration ofthe random primer is 6 ; determining the nucleotide sequence of the obtained DNA fragments; and designing a DNA probe used for detecting a DNA fragment obtained in the above step on the basis of the nucleotide sequence of the DNA nts.
[Claim 2] The method for ing a DNA probe ing to claim 1, wherein DNA fragments are obtained from a plurality of different genomic DNAs with the use of the random primers and, on the basis of the nucleotide sequence of the DNA fragments, the DNA probe containing regions different between the genomic DNAs is designed.
[Claim 3] The method for producing a DNA probe according to claim 1, wherein the nucleotide sequence of the DNA fragment is ed with a known nucleotide sequence and the DNA probe containing a region different from that of the known nucleotide sequence is designed.
[Claim 4]
[Claim 5]
[Claim 6] AMENDED SHEET Etinteclzgtsﬁﬁgglﬁ iEQIﬂP